Dormant tumors circumvent tumor-specific adaptive immunity by establishing a Treg-dominated niche via DKK3

Approximately 30% of breast cancer survivors deemed free of disease will experience locoregional or metastatic recurrence even up to 30 years after initial diagnosis, yet how residual/dormant tumor cells escape immunity elicited by the primary tumor remains unclear. We demonstrate that intrinsically dormant tumor cells are indeed recognized and lysed by antigen-specific T cells in vitro and elicit robust immune responses in vivo. However, despite close proximity to CD8+ killer T cells, dormant tumor cells themselves support early accumulation of protective FoxP3+ T regulatory cells (Tregs), which can be targeted to reduce tumor burden. These intrinsically dormant tumor cells maintain a hybrid epithelial/mesenchymal state that is associated with immune dysfunction, and we find that the tumor-derived, stem cell/basal cell protein Dickkopf WNT signaling pathway inhibitor 3 (DKK3) is critical for Treg inhibition of CD8+ T cells. We also demonstrate that DKK3 promotes immune-mediated progression of proliferative tumors and is significantly associated with poor survival and immunosuppression in human breast cancers. Together, these findings reveal that latent tumors can use fundamental mechanisms of tolerance to alter the T cell microenvironment and subvert immune detection. Thus, targeting these pathways, such as DKK3, may help render dormant tumors susceptible to immunotherapies.


Introduction
Multiple therapies now exist to treat different molecular subtypes of breast cancer (BC), leading to steady improvement in survival over the past two decades (1).Despite these successes, many survivors (approximately 30%) will eventually experience locoregional or metastatic recurrence, even when there was no clinical evidence of disease after initial therapy (2,3).Among solid tumors, BC has a propensity for delayed relapse with distinct patterns of recurrence based upon subtype.Those with triple negative breast cancer (TNBC), defined by lack of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), are particularly at risk of distant recurrence with a shorter window than other subtypes (33.9% vs 20.4%; 2.6 vs 5 years respectively) (4).In contrast, ER+ tumors can recur up to decades after treatment of the primary tumor and have a consistent risk of recurrence over time (4,5).Regardless, the time between remission and relapse offers a critical window to eliminate residual tumor cells before developing resistance mechanisms that make recurrent tumors exceedingly challenging to treat.This phenomenon of delayed relapse, often described generally as tumor dormancy, is largely attributable to residual tumor cells that enter a state of quiescence or minimal proliferation until some other condition for growth is attained (6).To date, multiple mechanisms help explain how these cells enter and exit quiescence.However, relatively little is known regarding their function during the intervening period.Though they are mostly non-proliferative, dormant cancer cells actively communicate with the local stroma to alter the microenvironment and support their own survival (7,8).Thus, understanding the intrinsic biology of residual, dormant tumor cells is necessary if attempting to eliminate them before recurrence.
The advent of immunotherapy has highlighted the role of immune cells in an evolving tumor, even during dormancy.Organ transplantation provided early evidence that the immune system prevents tumor outgrowth when occult tumors from donated organs began growing in the context of immunosuppressed recipients (9).Further, direct evidence that tumor-specific T cells prevent metastatic outgrowth was also recently described in BC (10,11), although how residual, dormant tumor cells are not eliminated remains in question.A potential explanation may be that fundamental, developmental mechanisms of tolerance, such as those present at the maternalfetal interface or during mammary involution, are co-opted to prevent complete tumor elimination (12).While unclear, if immunotherapy is to provide a lasting cure for patients, these overarching processes must be better understood.
Herein, we demonstrate that dormant tumors are recognizable by T cells, but manage to persist in the presence of activated tumor antigen-specific CD8+ T cells.Our studies reveal that dormancy-competent tumor cells maintain a hybrid epithelial/mesenchymal (E/M) or mammary progenitor-like state in vitro and in vivo.To survive long term, these cells induce a CD4/Treg shifted T cell profile via secreted factors and we identify the Wnt pathway antagonist, Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3), as a crucial mediator of this effect.Together, the data presented here provide insight into protective barriers that prevent elimination of dormant tumors and lay the foundation for new targets that may be combined with current immunotherapies to provide more durable responses for patients.

D2.1 dormancy is immune-independent
The D2 series of cells, consisting of D2A1, D2.OR, and D2.1 cell lines that arose from D2 hyperplastic alveolar nodules in female BALB/c mice, were used to investigate immunity in tumor dormancy (13,14).Each cell line is reported to equally extravasate into the lung parenchyma, but only D2A1 rapidly proliferates while D2.OR and D2.1 remain dormant in the metastatic setting (13,15,16).Tumor behavior in the presence or absence of adaptive immunity was first determined by implanting these cells in the mammary fat pad (MFP) of BALB/c (immunocompetent) or SCID-beige (deficient in T and B cells and defective NK cells) mice.Both D2A1 and D2.OR tumor growth was significantly delayed in the presence of adaptive immunity (Figure 1A, B).In contrast, D2.1 tumors were unaffected by T, B, and NK cells (Figure 1C).
However, when cultured in vitro D2A1 and D2.1 cells proliferate at approximately the same rate until confluence, at which point D2.1 cells become contact inhibited resulting in slowed growth (Supplementary Figure 1A, B).To validate the dormant phenotype, D2.1 cells were stably transduced with eGFP by lentiviral vectors and proliferation was assessed in resultant tumors by Ki67 immunofluorescence (IF) at 4 or 12 weeks post MFP implantation in BALB/c animals.
Staining revealed that eGFP+ D2.1 tumor cells were minimally positive for Ki67 at 4 weeks, but had significantly elevated Ki67 expression by 12 weeks (Figure 1D).Co-injection of a 1:1 mix of D2A1 and D2.1 cells also resulted in approximately equivalent tumor growth as D2A1 cells alone, suggesting dormant cells do not induce quiescence in otherwise proliferative cells (Supplementary Figure 1C).Collectively, these results demonstrate that D2.1 tumor cells exist as a separate, intrinsically dormant population, unaffected by immune status, that have the eventual capacity to develop into proliferative tumors.

Dormant tumors are immunologically protected from infiltrating T cells
Because D2.1 tumor persistence through the dormant phase was unaffected by adaptive immunity, cell surface expression of Major Histocompatibility Complex class I (MHC-I), Programmed death-ligand 1 (PD-L1), and CD47 was investigated as potential mechanisms for dormancy-mediated immune evasion (17,18).Notably, MHC-I (Figure 1E; Supplementary Figure 1D), PD-L1, and CD47 (Supplementary Figure 1E, F) are expressed similarly on D2A1, D2.OR, and D2.1 cells, suggesting that these are unlikely to mediate differences in immune resistance.As all D2 lines express comparable cell surface MHC-I, loss of eGFP expression after in vivo implantation was used as a readout for antigen-specific immune selection (Figure 1F) (19).
While many D2A1 tumor cells resisted eGFP-specific selection, D2.OR cells displayed the highest sensitivity with almost complete loss of eGFP (Figure 1G).In contrast, the majority of D2.1 cells maintained eGFP after implantation in immune competent, non-eGFP tolerant BALB/c mice.
These results are consistent with the recent finding that GFP-specific T cells (Jedi) are unable to eliminate quiescent sub-populations of mammary tumors despite MHC-I expression (20), and suggest that dormant tumors can be effectively shielded from antigen-specific immunity and selection by other means.

D2.1 dormant tumor cells have a hybrid E/M phenotype and are enriched in mammary progenitor genes associated with late recurrence in humans
Due to the striking phenotypic differences in vivo between dormancy-competent D2.1 cells and proliferative counterparts we initially performed RNAseq on D2A1 and D2.1 cell lines.The top significantly enriched pathways in D2.1 cells were largely associated with cellular movement and extracellular matrix (ECM) organization with a significant downregulation of metabolic pathways involved in DNA and RNA processing (Figure 2A; Supplementary Figure 2A).Notably, D2.1 cells expressed higher transcript levels of multiple mammary epithelial makers genes (e.g.Krt8, Krt14, Krt18, Itgb4, Epcam, Cdh1) and genes formerly associated with mammary progenitor cells (e.g.Sox9, CD74, Id4, Nrg1, Ptn, Cd200) (Figure 2B).Genes that regulate epithelial to mesenchymal transition (EMT) and metastasis were also upregulated, including genes associated with ECM interaction (Itga5, Sparc, Mmp9, Cd44, and Fn1) as well as many transcriptional regulators of EMT (Twist2, Snai1, Snai2, Notch1) (Figure 2B).Subsequent quantitative PCR confirmed the hybrid E/M phenotype of D2.1 cells which expressed high levels of epithelial and mesenchymal genes compared to both D2A1 and D2.OR cells (Supplementary Figure 2B).The ERα gene Esr1 and the ERα target gene Greb1 were also expressed in D2.1 cells (Supplementary Figure 2C)an interesting corollary as human ER+ tumors generally display the highest degree of dormancy (5,21).
In accordance with an overall mammary progenitor-like expression profile, D2.1 cells were found to be CD44 hi CD24 low/neg compared to D2.OR and D2A1 cells by flow cytometry, with D2.OR cells having the most CD24 expression (Supplementary Figure 2D) (22,23).The presence of increased epithelial and mesenchymal genes could also reflect the described "hybrid Epithelial/Mesenchymal (E/M)" state associated with surface CD44 and CD104 expression (24,25).Interestingly, surface expression of CD44 and CD104 revealed largely non-overlapping populations between D2A1, D2.OR, and D2.1 cells (Supplementary Figure 2E).Because D2.OR tumors are immune-sensitive (Figure 1G), overt tumors were often rejected in the MFP of BALB/c animals.However, small, residual nodules that re-emerged after approximately 100 days phenotypically resembled D2.1 cells based upon CD44/CD104 expression (Supplementary Figure 2F).Altogether, these data indicate that D2.1 cells have hybrid E/M phenotype at baseline which has been associated with metastasis, dormancy, and immunosuppression (24)(25)(26)(27), and may represent a particular sub-population that can re-emerge after immune rejection of the tumor at large (as from D2.OR tumors).

D2.1 tumor phenotype is maintained throughout progression
Bulk RNAseq was also performed on MFP D2A1 and D2.1 tumors from both BALB/c and SCID beige mice to evaluate phenotypic differences and changes from the selective pressure of adaptive immunity (Supplementary Figure 3A).Overall, D2A1 or D2.1 tumors were largely similar whether in BALB/c or SCID beige mice (Supplementary Figure 3B, C).The hybrid E/M and mammary progenitor expression pattern was maintained long-term in D2.1 tumors (Figure 2C, D) (28).Furthermore, D2.1 tumors were enriched for a genetic profile of late recurrence we previously developed from human BC samples (29), as well as an independent profile of late distant metastasis also generated from human data (30)(Figure 2E, F; Supplementary Table 1).
Pathway analysis also revealed that even later stage D2.1 tumors displayed a less proliferative phenotype when compared to D2A1 tumors in both Balb/c and SCID beige animals (Supplementary Figure 3D, E) with upregulated Cdkn1b (p27; a common indicator of tumor dormancy (20,31)) and reduced expression of genes associated with proliferation (Supplementary Figure 3F).Somewhat surprisingly, GO analysis revealed that eight of the top ten upregulated pathways in D2.1 tumors specifically related to immune activation (Figure 2G), and while general markers of T cells (Cd3d, Cd3e, Cd3g) and cytotoxic T cells (Cd8a, Cd8b1) were associated with tumors from all BALB/c animals, T regulatory cell (Treg) genes (Ctla4, Foxp3) were only associated with D2.1 tumors in immunocompetent animals (Supplementary Figure 3G).Subsequent CIBERSORT analysis indicated increased CD4 T follicular helper cells, CD8 cells, Tregs, and Naïve B cells in D2.1 compared to D2A1 tumors (Figure 2H) despite the possibility that these immune cells were less functional (Figure 2I).
As the Myc oncogene was significantly upregulated in D2A1 tumors (Figure 2B,C; Supplementary Figure 4A), we speculated that the dormant phenotype of D2.1 cells could be reversed by Myc stimulated proliferation.To test this, we expressing a stabilized form of Myc (T58A) (32) in D2.1 cells (Supplementary Figure 4B).While Myc-T58A expression enhanced proliferation in vitro and in SCID beige mice it did not translate to enhanced tumor burden in immune competent BALB/c mice (Supplementary Figure 4C,D).This suggests pro-proliferative signaling in tumors is insufficient to counteract stong immune-editing of proliferative cells, and that a period of dormancy followed by growth is potentially a necessary stage for immune escape in these populations.

Dormant mammary tumors have high levels of infiltrating Foxp3+ T regulatory cells
In light of the immune-independent nature of D2.1 latency the presence of immune-related genes in D2.1 tumors was striking.Therefore, D2.1 cells were orthotopically implanted and collected after 35 days for immune analysis, and D2A1 tumors were either collected early at the same final size as D2.1 tumors (14 days post-implantation) or after the same total duration (35 days post-implantation) to ensure that potential differences were not merely due to tumor burden (Figure 3A, B).Interestingly, flow cytometry revealed that D2.1 tumors indeed contained significantly more total T cells than D2A1 tumors of the same volume or time in vivo (Figure 3C).
To ascertain the spatial relationship of T cells with tumor cells and determine if differences in T cell profiles were maintained through tumor progression, D2.1 tumors were also collected at a later stage (100 days post-implantation) (Supplementary Figure 5B).Staining for CD4+ and CD8+ T cells revealed that both collected at the tumor/stromal interface in size-matched D2A1 tumors (Figure 3H; a feature observed in approximately 26% of TNBCs (33)).However, endpoint D2.1 tumors were highly infiltrated with both CD4+ and CD8+ T cells (Figure 3I) resulting in a significantly increased interior T cell density in comparison to similarly sized D2A1 tumors (Figure 3J, K).Furthermore, D2.1 tumors harbored a significant population of infiltrating FoxP3+ cells, which likely represents the same CD4+ FoxP3+ Treg population observed via flow cytometry based upon multicolor IHC (Figure 3L; Supplementary Figure 5C).No correlation in CD8+ cell density was observed collectively across D2.1 tumors of multiple timepoints/sizes (Figure 3M), however Foxp3+ cell infiltration was significantly correlated with early, smaller tumors (Figure 3N).Thus, dormant tumors do not necessarily restrict T cell infiltration even after beginning to proliferate, indicating that changes in T cell function may be more relevant than proximity.
Additionally, these data suggest that Treg induction is an early and necessary event to establish the dormant tumor niche for long-term survival.

Tumor/antigen-specific CD8+ cells do not prevent dormant tumor progression in vivo
Primary mammary tumors were recently reported to induce CD8+ T cells (CD39+ PD-1+) that systemically control metastatic dormancy in the lungs in the 4T07 model (10).Contrastingly, analysis of MFP D2A1-eGFP and D2.1-eGFP tumors (Supplementary Figure 5E) revealed that D2A1 tumors contained significantly more CD39+ PD-1+ CD8+ T cells than D2.1 tumors, with a similar trend seen systemically in the spleens of tumor-bearing animals (Figure 4A).Absent a strong, de novo tumor-specific T cell response, we next investigated if D2.1 persistence could be overcome with potent and systemic antigen-specific T cells.To exclude the role of the tumor microenvironment, killing assays were initially performed in vitro to assess the cytotoxic effect of antigen-specific CD8+ T cells against D2.1 and D2A1 cells.Just eGFP Death Inducing (Jedi) T cells, which express a H-2Kd restricted T cell receptor (TCR) for GFP200-208 (Figure 4B) (34), were activated/expanded ex vivo and cultured with D2A1 or D2.1 eGFP cells.Both D2A1 and D2.1 cells were equivalently lysed by Jedi cells thereby verifying that both are susceptible to direct killing by activated Jedi CD8+ T cells (Figure 4C, D).Adoptive transfers were then performed to determine if externally activated antigen-specific CD8+ T cells could prevent/limit eventual D2.1 tumor outgrowth.Activated Jedi cells were injected intravenously into naïve C57Bl/6 × BALB/c F1 mice and two days later D2A1 or D2.1 cells expressing eGFP were implanted into the MFP (Figure 4E).While D2A1 tumor outgrowth was completely prevented by Jedi T cells compared to control T cells (Figure 4F), D2.1 tumors were unaffected by Jedi T cells in comparison to controls (Figure 4G).These results indicate that even highly activated antigen-specific CD8+ T cells are incapable of preventing establishment and eventual growth of dormant D2.1 tumors in vivo.

D2.1 cells secrete factors that induce CD4+ and Foxp3+ T regulatory cells
Increasing evidence suggests that epithelial/mesenchymal plasticity, as observed in D2.1 cells, is associated with an overall immunosuppressive microenvironment (27).However, because CD8+ T cells were distinctly present in but unable to eliminate latent tumors, we investigated if tumor cells themselves altered T cell function directly.The effect of the tumor secretome on CD8+ T cell activation/proliferation was initially to be tested by culturing splenocytes from transgenic Jedi mice with tumor-derived conditioned medium (CM) (35).Whole Jedi splenocytes were stained with Cell Trace dye and stimulated with GFP200-208 peptides/anti-CD28 antibodies in the presence of D2A1 or D2.1 CM for 3 days (Figure 5A).Overall, D2.1 CM resulted in significantly more T cells than D2A1 CM (Figure 5B), including both CD8+ (which was stimulated via peptide) and CD4+ cells (Figure 5C).Cell Trace staining indicated that CD8+ T cells indeed underwent more divisions in D2.1 CM compared to D2A1 CM (Figure 5D), however CD4+ T cells displayed more significantly enhanced proliferation in D2.1 CM in comparison to D2A1 CM (Figure 5E).
Further experiments revealed a significant increase in Tregs within the CD4 population (Figure 5F), ultimately resulting in a decreased CD8:Treg ratio in D2.1 CM compared to D2A1 (Figure 5G).Thus, these data suggest that the disparity between D2A1-and D2.1-induced T cell responses in vivo are likely more attributable to dormant tumor cell promotion of CD4+ Treg proliferation and/or differentiation via soluble factors.

T regulatory cells protect D2.1 tumors
Because CD8+ T cell functionality was ostensibly restricted in D2.1 tumors with an associated increase in Tregs, we estimated that Tregs were a more central determinant of immunosuppression in the dormant tumors.Given the ability of anti-CTLA4 IgG2A/B antibodies to deplete intra-tumoral Tregs (36,37), we first treated mice bearing D2.1 tumors bi-weekly with anti-CTLA4 (clone 9D9, 200ug/mouse) or isotype controls upon reaching ~150 mm 3 .This treatment significantly reduced tumor growth (Figure 5H; Supplementary Figure 6A) with a modest reduction of intra-tumoral Tregs (although total T cell levels were unchanged) (Supplementary Figure 6B).While these results suggested the importance of Tregs in dormant tumors, anti-CTLA4 antibodies can alter co-stimulation, thus we wanted to validate these findings in a more specific Treg model.We therefore implanted D2.1 cells into FoxP3-DTR mice, in which Tregs can be ablated through Diptheria Toxin (DT) administration (Supplementary Figure 6C).
Tumor bearing animals were treated with DT every four days beginning at an early time point (day 35) or late time point (day 70) with a total of five doses each (Figure 5I).Notably, both early and late DT treatment resulted in significant reduction of tumor growth (Figure 5J; Supplementary Figure 6D, E).Late DT treated and control tumors were analyzed upon euthanasia which showed an increase in infiltrated T cells (Supplementary Figure 6F), including an increase in activated CD39+ PD-1+ CD8+ T cells.Altogether, these data indicate that despite high CD8+ T cell infiltration, the concomitant presence of Tregs in D2.1 tumors restricts anti-tumor immunity and enables tumor persistence.

DKK3 is crucial for D2.1 tumor persistence
Knowing that tumor-derived secreted factors were sufficient to regulate the T cell landscape we further investigated potential soluble mediators of this effect.The hybrid E/M and mammary progenitor-like signature of D2.1 tumors was typified by high expression of Dickkopfrelated protein 3 (DKK3), a Wnt signaling modulator that is preferentially expressed by CD44+ CD24-epithelial stem/progenitor cells within the human mammary gland and is generally enriched across stem cell or "immune privileged" niches, making it an attractive candidate (Figure 6A) (38,39).Interestingly, DKK3 expression was even greater in D2.1 tumors in immune-competent animals but not D2A1 (Figure 6B, C).Elevated DKK3 expression in D2.1 cells compared to D2A1 or D2.OR cells was confirmed via quantitative PCR (Supplementary Figure 7A) and its function was assessed after stable shRNA knockdown (k/d) in D2.1 cells (Supplementary Figure 7B, C).
Upon MFP implantation, shDKK3 D2.1 tumors displayed significantly reduced growth in vivo compared to shScramble controls with the majority being completely rejected (5/8 mice; Figure 6D; Supplementary Figure 7D).Subsequent analysis of tumor cells after expansion ex vivo confirmed that non-rejected shDKK3 tumors regained DKK3 expressionlikely explaining persistence of these tumors (Supplementary Figure 7E).To validate these results, two independent Crispr DKK3 knockout (KO) D2.1 clones were generated (Supplementary Figure 7F, G).When implanted into immunocompetent animals DKK3 KO cells were completely rejected (Figure 6E) whereas in SCID beige animals DKK3 KO cells yielded tumors equivalent to control cells (Figure 6F).Thus, DKK3 expression indeed appears to be a necessary component for immune-escape and long-term survival during the dormant phase.

Tumor-derived DKK3 regulates Tregs to inhibit CD8+ T cell function
The mechanistic underpinnings of DKK3-mediated immune evasion were further validated ex vivo by stimulating isolated CD8+ Jedi T cells mixed with antigen presenting cells (APCs) in D2.1 shScramble or shDKK3 CM.Unexpectedly, no difference in CD8+ T cell proliferation was detected between D2.1 shDKK3 CM compared to control (Figure 6G).However, additional experiments using a mix of CD8+ Jedi cells, CD4+ cells, and APCs revealed significantly increased total T cells in shDKK3 CM compared to shScramble (Figure 6H) with increased CD8+ T cell proliferation, suggesting an indirect effect of DKK3 on CD8+ T cells.Consistent with this observation, the CD4+ T cell fraction was significantly reduced in DKK3 silenced D2.1 CM (Figure 6I) with a decrease of CD4+Foxp3+ Tregs (Figure 6J) yielding an increased CD8:Treg ratio (Figure 6K).Similar results were also observed when using CM from DKK3 Crispr KO cells (Supplementary Figure 8A-C).As a proxy for immune activation, we also assessed the level of IFNγ in media by ELISA and found that DKK3 k/d resulted in increased IFNγ compared to control D2.1 CM and comparable to culture in D2A1 CM (Figure 6L).Altogether, these experiments support the previous data and implicate CD4+ T cells, particularly Tregs, as a target of D2.1 regulation via secretion of DKK3, with secondary effects on CD8+ cells.

DKK3 is associated with poor survival and immune-evasion in human BC
Given the experimental evidence of DKK3-modulated immune function, DKK3 was assessed in BC datasets obtained from clinical samples to determine relevance of this pathway in humans.In BC, we found that high Dkk3 expression was significantly correlated with poor survival in Basal and Luminal B tumors in both the KMPlotter and TCGA datasets (Figure 7A,B).
Interestingly, Dkk3 was inversely correlated with genes indicative of an anti-tumor immune response (e.g.Cd4, CD8a, Gzma, Gzmb, Ifng) in Basal BC across datasets, a pattern not observed with family members Dkk1 or Dkk2 (Figure 7C; Supplementary Table 2).Furthermore, Dkk3 was positively correlated with the ectonucleotidases Nt5e (CD73) and Entpd1 (CD39), which are highly expressed by Tregs and critically mediate peripheral tolerance (40), and negatively correlated with the proliferation marker Mki67 (Figure 7C).Expression of Dkk3 was also higher in primary tumors of BC patients that presented with metastasis, even in ER+ tumors which have higher Dkk3 at baseline (Figure 7D, Supplementary Figure 9A).Single-cell RNA-seq analysis of human breast cancers revealed that Dkk3 is expressed by multiple cells within the breast microenvironment, including both normal and cancer epithelial cells, including TNBC cells (Supplementary Figure 9B,C).Thus, in human BC both the stroma or tumor cells themselves likely contribute to DKK3 in the microenvironment (41).

DKK3 promotes immune evasion in rapidly progressing models of BC
To directly assess the role of DKK3 in suppressing anti-tumor immunity, we utilized lentiviral vectors to stably overexpress DKK3 in D2.OR cells, which we found to be highly sensitive to adaptive immune responses (Supplementary Figure 10A; Figure 1G).While DKK3 expression yielded no significant difference in cellular proliferation in vitro, it did elicit a CD44/CD104 profile similar to D2.1 cells or residual D2.OR tumors (Supplementary Figure 10B,    C).Upon MFP implantation in immune competent hosts, DKK3 expression provided a robust engraftment and survival advantage compared to control empty vector D2.OR tumors, which were rejected after stable integration of the puromycin xenoantigen (Supplementary Figure 10D).
Prior reports suggest that DKK3 enables MHC-I mismatched transplantation of embryoid bodies (42), therefore to test the possibility that DKK3 protected against foreign antigen-specific immunity, a vector containing eGFP in addition to DKK3 was lentivirally transduced into D2.OR cells which were subsequently transplanted into the MFP of BALB/c mice.As with puromycin expressing D2.OR cells, we found that D2.OR cells expressing eGFP alone were completely rejected while parental D2.OR cells formed tumors (Figure 7E).However, D2.OR cells expressing both eGFP and DKK3 successfully engrafted, although at a diminished rate compared to parental cells (Figure 7E).Moreover, examination of infiltrating T cells revealed an increase in Tregs compared to parental tumors (Figure 7F) and a systemic decrease in effector CD8+ cells in the spleen (Figure 7G).Together, these data suggest that DKK3 enables antigen-specific protection in otherwise immune-sensitive tumors and supports the role for DKK3 in Treg generation.Congruent results were observed when expressing DKK3 in highly proliferative D2A1 cells (Supplementary Figure 10F).We found that DKK3-expressing D2A1 cells grew equivalently whether implanted into the MFP of Balb/c or SCID beige mice whereas control tumors were substantially delayed in Balb/c animals (Supplementary Figure 10G), and ELISA of serum at time of euthanasia confirmed that DKK3 expression was maintained (Supplementary Figure 10H).By the end of the experiment, effector CD8+ T cells were significantly reduced in the tumor (Supplementary Figure 10I) and large alterations in splenic T cells were detected (Supplementary Figure 10J), with T cells were shifted towards a naïve (CD44 -CD62l + ) phenotype.Less anti-eGFP antibodies were also present in the serum of mice bearing DKK3expressing D2A1 tumors when accounting for tumor volume (Supplementary Figure 10K).In sum, these in vivo data reveal that DKK3 can restrict adaptive anti-tumor immunity during multiple stages of tumor progression.

Discussion
Tumor dormancy and re-emergence depend on both tumor-intrinsic and -extrinsic mechanisms that are slowly being uncovered.Our study does not address how tumors enter and exit dormancy (although others have recently shed light on dormancy in D2 cells( 7)), however we describe an avenue by which intrinsically dormant and tumor populations evade adaptive immunity to eventually form progressive disease.This is achieved via early initiation of a CD4+/Treg skewed T cell response, and we provide evidence that the tumor-derived Wnt modulator DKK3 is critical to this process.We also demonstrate that DKK3 is essential for tumor survival during the latent phase and generally protects tumors from antigen-specific CD8+ T cell immunity.Finally, our analysis suggests that DKK3 is a relevant target in BC, particularly in TNBC and Luminal B tumors.An important determinant of recurrence is the duration that CD8+ T cells maintain in their cytotoxic dominance against proliferative tumor cells (43,44).Evidence suggests that CD39+ PD-1+ CD8+ T cells represent a locally-induced, tumor-reactive population that can also prevent metastatic outgrowth in BC and others (10,45).However, our studies revealed that dormant tumor cells prevent generation of this CD8+ T cell population compared to more proliferative tumors.Critically, we demonstrate that activated tumor antigen-specific CD8+ T cells were unable to eliminate these latent tumors, but that targeting Tregs specifically allowed the generation of more effective anti-tumor responses, suggesting a primacy of Tregs in protecting dormant cancer cells.
The degree to which dormant tumors are targetable by CD8+ T cells in general also remains elusive.It is commonly understood that MHC-I downregulation in dormant/disseminated tumor cells can promote immune escape (17,46,47), although our data suggest the importance of microenvironmental avenues of immune evasion as opposed to being "invisible" to adaptive immunity.A growing body of literature indicates that MHC-I/antigen presentation is highly dynamic (43,48), supportive of other studies that have found no differences in antigen presentation pathway genes or MHC-I expression in dormant BC cells (20).Collectively, these studies suggest that, although they are in fact detectable by CD8+ T cells, many dormant BC tumor cells rely on other means to escape elimination.
While Tregs are generally important for tumor immune evasion (49), a definitive role in protecting dormant tumor cells remains elusive (50).Clinical studies suggest that Tregs are specifically associated with late relapse (>5 years) and poor metastatic survival in BC (51,52).
We found direct evidence that tumor-derived DKK3 was able to induce Treg differentiation in vitro which ultimately resulted in decreased CD8+ T cell function.While others have reported direct effects of DKK3 on CD8+ cells (42,53,54), we found the effect was most prominent on CD4+ T cells/Tregs.Recently, DKK3 was described to regulate downstream Wnt/β-catenin genes Tcf7, which encodes T cell factor-1 (TCF-1), and Lef1 (encoding Lymphoid Enhancer Binding Factor 1), in CD4+ cells to coordinate IFNγ secretion by a yet to be determined receptor (55).Canonical Wnt signaling also inhibits Treg activity (56), therefore suppression of Wnt by tumor-derived DKK3 may mediate CD4 differentiation towards Tregs.Additionally, other studies support a role for DKK3 in concert with other factors in the extracellular milieu, such as TGF-β (57-60).The TGF-β pathway is especially relevant for Treg function (61), and because D2.1 cells also expressed more Tgfb1 we speculate that this may be an important connection to delineate the contextual function of DKK3 on T cells in addition to Wnt.
Currently the role of DKK3 in cancer is poorly understood with both inhibitory and beneficial functions being reported in tumors (41,(62)(63)(64)(65)(66)(67)(68).In our hands DKK3 did not directly alter tumor cell proliferation, but instead showed tumor-promoting effects when in contact with the immune system.This is consistent with recent studies in pancreatic tumors that demonstrated increased T cell accumulation in DKK3 KO mice and others showing greater tumor rejection with enhanced CD8+ T cell infiltration into tumors when mesenchymal stem cells were DKK3 deficient (54,69).More generally, DKK3 was identified as necessary for generating antigen-specific, tolerant CD8+ T cells in vivo and could suppress T cell proliferation in vitro (53).In an autoimmune context, DKK3 produced locally by stromal cells in the skin was found to reduce self-antigen induced experimental autoimmune encephalomyelitis (EAE) symptoms in a CD4+ T celldependent fashion (42,70).As such, our results further elucidate an ability of DKK3 to restrain adaptive immunity in normal and pathological conditions through the induction of Tregs, although the exact molecular drivers of this process will need further clarification.
Along with other secreted factors, other cell types in specific niches may play a critical role in DKK3 expression and function.Our results were obtained in an orthotopic site (i.e. the MFP) with tumor-derived cytokines.As mentioned previously, within the human mammary gland Dkk3 is one of the most upregulated genes in CD44+CD24-(stem) vs CD44-CD24+ (differentiated) cells (38), which mimics the phenotype of D2.1 vs D2A1/D2.OR cells.However, other cells within the local tumor microenvironment and certain disseminated niches may also be important contributors of DKK3.Interestingly, DKK3 is commonly expressed by stromal cells in "immune privileged" and stem cell niches in the brain, eye, pancreas, liver, and bone marrow (39,(71)(72)(73)(74)(75)(76).
The trophectoderm, which induces immune tolerance at the maternal-fetal interface, in part by recruiting and/or inducing Tregs, is also enriched for DKK3 expression (77)(78)(79).Thus, DKK3 may be a component of larger mechanisms that maintain stem cell niches and prevent aberrant immune activation against these critical populations, which we speculate is driven by the plastic E/M phenotype (12) and the fact that DKK3 can prevent MHC-I mismatched embryonic body rejection (42) supports this notion.Currently, immune checkpoint blockade (ICB) remains largely unsuccessful in hormone receptor positive BC (which are most likely to go dormant) due to low T cell infiltration and an immunosuppressive stroma (80).Although pancreatic cancer is similarly resistant to ICB, combined anti-CTLA4 + anti-DKK3 antibodies showed synergistic anti-tumor responses in a pancreatic cancer model (54).Although ICB has been approved for certain TNBCs, great interest also remains in combinatorial therapies that target alternative pathways to boost ICB response (81).Therefore, targeting DKK3 alongside other immunotherapeutic strategies may help break alternative suppressive barriers to eliminate dormant, residual tumor cells and more aggressive cells alike.

Lentiviral production and infection
All lentiviral vectors were produced in 293T cells using second-generation packaging plasmids and previously described techniques and viral stocks were concentrated by ultracentrifugation.Viral stocks were added to medium containing 8 µg/mL polybrene and cells were incubated for 48 hours before sub-culturing.Transduced cells were selected via puromycin (shRNA knockdown, CRISPR knockout) or sorting for eGFP+ cells.Single cell clones were generated by limited dilution in 96 well-plates and subsequent culture of wells containing a single colony.

Orthotopic implantation
Tumor cells in sterile PBS were implanted into the fourth inguinal mammary fat pads under general anesthesia.Tumors were monitored by caliper and volume was calculated using the formula volume = (length × width 2 ) ÷ 2.

Killing assays
D2A1 or D2.1 target cells expressing eGFP-Luc were plated at 2,000 cells/well in a 96 well plate and allowed to attach overnight at 37°C.The following day, serial dilutions of effector Jedi T cells (activated as for adoptive transfer) were added to each well in quadruplicate and plates were incubated overnight at 37°C.Cells were lysed with a Triton-x 100 buffer and luciferase activity was measured using a Veritas microplate luminometer (Turner Biosystems).The fraction of remaining cells in each well was normalized to the average signal of control wells that did not receive Jedi cells.

Conditioned medium (CM) harvest
Tumor cells were seeded in complete medium at 5×10 3 cells/mm 2 in a 10 cm dish and allowed to attach before changing medium to low-glucose DMEM containing 1% FBS.Cells were cultured for 48 hours at which point conditioned medium was collected, centrifuged to remove debris, passed through a 0.45µm filter, and stored at 4°C until use.

T cell activation/proliferation assays
CD4/CD8 T cell isolation: Splenocytes were resuspended in EasySep buffer (Stemcell Technologies) at 10 6 cells/mL and CD4 cells were isolated with EasySep Mouse CD4+ T Cell Isolation Kit (Stemcell Technologies) or CD8+ cells were isolated using the EasySep Mouse CD8+ T Cell Isolation Kit (Stemcell Technologies) according to the manufacturer's protocol.
CellTrace staining: Single cells (whole splenocytes or CD8+ cells only) were resuspended in PBS at 10 6 cells/mL.Cells were stained with 1 µL/mL (after reconstitution in 20 µL DMSO) CellTrace Far Red (Inivtrogen) for 20 minutes at 37°C with gentle mixing every 5 minutes.Cell suspension was diluted 5× with complete medium and incubated at room temperature for 5 minutes and washed 1× with medium.

Immunohistochemistry (IHC)/Immunofluorescence (IF)
Formalin-fixed, paraffin-embedded tissue sections were deparaffinized with xylene and rehydrated through graded concentrations of ethanol and distilled water.Epitope retrieval was performed in a Retriever 2100 (Aptum Biologics) with R-Buffer A (Electron Microscopy Sciences).IHC: Endogenous peroxidases/phosphatases were quenched with BLOXALL blocking solution (Vector) and tissues were blocked with Animal-Free Blocker R.T.U.(Vector).Sections were probed with primary antibodies (for a complete list see Supplementary Table 4) overnight at 4°C, washed with PBS, and incubated with the appropriate ImmPRESS polymer detection reagent (Vector) for 30 minutes at room temperature.Visualization was performed by incubation with 3,3′-diaminobenzidine (DAB) (Vector), ImmPACT Vector Red (Vector), or a Green HRP staining kit (Novus), For triple IHC, a second round of retrieval was performed with R-Buffer A after developing the first round of HRP and AP stains.Tissues were counterstained with Gill No.3 Hematoxylin (Sigma), cover-slipped, and imaged on an Olympus IX73 inverted microscope with a 40x objective.Infiltrated T cells were enumerated on 5 random fields of view per sample using ImageJ software.
IF: Tissues were blocked with Animal-Free Blocker R.T.U. and incubated in primary antibodies overnight at 4°C.Tissues were washed with PBS, and secondary staining was performed for 1 hour in the dark at room temperature with the appropriate fluorophore-conjugated antibody.Tissues were counterstained with DAPI and cover-slipped with VECTASHIELD Vibrance (Vector) mounting media.Whole-slide images were collected using a Zeiss LSM880 confocal microscope.Ki67 staining analysis was performed using CellProfiler software.Single nuclei were segmented using the DAPI channel and the eGFP channel was used to create a binary mask to delineate between tumor and stroma.The number of Ki67+ nuclei within the eGFP mask was quantified and represented as a percent of the total nuclei within the eGFP mask.

Quantitative real-time PCR
RNA was isolated using Qiagen RNAeasy kits and reverse transcribed using iScript cDNA Synthesis Kit (Bio Rad) before performing quantitative PCR using Sso Advanced Universal SYBR Green Supermix (Bio Rad).Values were determined using the ΔΔCT method with actin as internal control.Primer pairs can be found in the supplementary materials.

Flow cytometry
Tumors were digested in serum-free medium using collagenase (1 mg/mL), DNAse (20 U/mL), and hyaluronidase (100 μg/mL) for 90 minutes at 37°C.Spleens were mechanically dissociated as before.Samples were washed with serum-containing medium, red blood cells were lysed with ACK buffer, and passed through a 40-µm cell strainer to generate single cell suspensions.For each condition 2×10 6 cells were stained at 4°C with Fc Block (CD16/32 clone 93; BioLegend), Live/Dead Fixable Dye (Invitrogen) and a combination of directly conjugated antibodies (for a complete list see Supplementary Table 4).Cells were fixed in 1% paraformaldehyde and analyzed on a BD LSR or Cytek Northern Lights flow cytometer.

Quantitative ELISA
Mouse IFNγ in T cell cultures was detected using the ELISA MAX Set from BioLegend according to the manufacturer's protocol.Anti-mouse DKK3 ELISA kits were purchased from RayBiotech and serum DKK3 was detected according to the standard protocol.

RNA-seq
Individual tumors were snap frozen and stored in RNAlater-ICE (Invitrogen) until RNA extraction.Total RNA was extracted using PureLink RNA mini Kits (Invitrogen) and DNA was removed via DNA-free DNA Removal kit (Invitrogen).RNA quality and concentration was determined using an Agilent 2100 Bioanalyzer.Whole transcriptome sequencing of cell lines and tumors was performed by Novogene on an Illumina NovaSeq 6000.Read alignment, quality control, differential expression analysis, and pathway analysis was performed by Novogene using the standard pipeline or GENAVi (84).CIBERSORT analysis was performed by TIMER2.0 using the immune estimation function (http://timer.cistrome.org/).Gene-set enrichment analysis (GSEA; version 4.2.3) was performed on DESeq2 normalized counts using the gene_set permutation type with Mammary stem/progenitor signatures (28) accessed via the MsigDB and previously published late recurrence or late distant metastasis signatures (30).

Clinical analysis
Survival and expression correlation analysis of select genes was performed using the

Statistical analysis
No statistical methods were used to pre-determine animal numbers and when appropriate animals were randomly assigned to treatment groups using the RANDBETWEEN function in       performed by two-tailed ttest.An outlier was identified for G (right; empty triangle) using the outlier analysis function in GraphPad Prism (ROUT method, q = 1%) and the p value displayed excludes the outlier (p = 0.1309 included).Error bars represent mean ± SEM.
Kaplan-Meier Plotter tool (Breast cancer, mRNA gene chip; https://kmplot.com/).Expression levels of Dkk3 (Affymetrix ID 214247_s_at) were split by upper and lower quartiles to compare relapse free survival (RFS) by PAM50 subtype.Overall survival (OS) data, PAM50 classification, and RNAseq log2 normalized counts for the TCGA-BRCA dataset was accessed via UCSC Xena Browser (https://xenabrowser.net/) and stratified based upon upper and lower Dkk3 quartiles for survival.METABRIC and the Metastatic Breast Cancer Project mRNA expression data and clinical characteristics were accessed via cBioPortal (https://www.cbioportal.org/).
Excel.Data were visualized and analyzed using GraphPad Prism v9 with each point representing a single mouse for in vivo experiments or technical replicates for in vitro experiments.In vitro experiments were performed at least twice with similar results and in vivo experiments were repeated with similar results or validated with complimentary experiments.Details of statistical analyses can be found in the figure legends and p values are displayed within the figures.P values ≤0.05 were considered significant.Study approvalAll animal studies were performed in accordance with Duke IACUC approval (protocol A043-23-02) and supervised/housed by the Division of Laboratory Animal Resources (DLAR).

Figure 1 :
Figure 1: The adaptive immune system does not impact tumor dormancy and long-term

Figure 2 :
Figure 2: D2.1 tumor cells are enriched in mammary progenitor genes and maintain a hybrid E/M profile in vivo associated with immune dysfunction

Figure 3 :
Figure 3: Dormant tumors and are highly infiltrated by T cells but induce a Treg-rich

Figure 7 :
Figure 7: DKK3 is associated with poor survival, decreased effector function, and metastasis in