KRASG12D drives immunosuppression in lung adenocarcinoma through paracrine signaling
Emily L. Lasse-Opsahl, Ivana Barravecchia, Elyse McLintock, Jennifer M. Lee, Sarah F. Ferris, Carlos E. Espinoza, Rachael Hinshaw, Sophia Cavanaugh, Marzia Robotti, Lily Rober, Kristee Brown, Kristena Y. Abdelmalak, Craig J. Galban, Timothy L. Frankel, Yaqing Zhang, Marina Pasca di Magliano, Stefanie Galban
Emily L. Lasse-Opsahl, Ivana Barravecchia, Elyse McLintock, Jennifer M. Lee, Sarah F. Ferris, Carlos E. Espinoza, Rachael Hinshaw, Sophia Cavanaugh, Marzia Robotti, Lily Rober, Kristee Brown, Kristena Y. Abdelmalak, Craig J. Galban, Timothy L. Frankel, Yaqing Zhang, Marina Pasca di Magliano, Stefanie Galban
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Research Article Oncology

KRASG12D drives immunosuppression in lung adenocarcinoma through paracrine signaling

Abstract

Lung cancer is the leading cause of cancer deaths in the United States. New targeted therapies against the once-deemed undruggable oncogenic KRAS are changing current therapeutic paradigms. However, resistance to targeted KRAS inhibitors almost inevitably occurs; resistance can be driven by tumor cell–intrinsic changes or by changes in the microenvironment. Here, we utilized a genetically engineered mouse model of KRASG12D-driven lung cancer that allows for inducible and reversible expression of the oncogene: activation of oncogenic KRASG12D induces tumor growth; conversely, inactivation of KRASG12D causes tumor regression. We showed that in addition to regulating cancer cell growth and survival, oncogenic KRAS regulated the transcriptional status of cancer-associated fibroblasts and macrophages in this model. Utilizing ex vivo approaches, we showed that secreted factors from cancer cells induced the expression of multiple cytokines in lung fibroblasts, and in turn drove expression of immunosuppressive factors, such as arginase 1, in macrophages. In summary, fibroblasts emerged as a key source of immune regulatory signals, and a potential therapeutic target for improving the efficacy of KRAS inhibitors in lung cancer.

Authors

Emily L. Lasse-Opsahl, Ivana Barravecchia, Elyse McLintock, Jennifer M. Lee, Sarah F. Ferris, Carlos E. Espinoza, Rachael Hinshaw, Sophia Cavanaugh, Marzia Robotti, Lily Rober, Kristee Brown, Kristena Y. Abdelmalak, Craig J. Galban, Timothy L. Frankel, Yaqing Zhang, Marina Pasca di Magliano, Stefanie Galban

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Figure 5

Identification of KRASG12D-dependent immunosuppressive secretome.

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Identification of KRASG12D-dependent immunosuppressive secretome. (A) Sc...
(A) Schematic depicting generation of LC3-547, an L-iKRAS cancer cell line from a murine lung tumor. (B) Representative Western blot depicting RASG12D and p53 protein expression in LC3-547 cells, which were cultured in dox-containing media for 24 hours prior to withdrawal of dox, and in A549 cells as controls. (C) Representative Western blot of RASG12D expression, p-ERK1/2, and vinculin in LC3-547 cells treated with 1 μM MRTX or 1 μM sotorasib (Soto) for 6 hours. (D) Depiction of the experimental outline for the collection of RNA and tumor-conditioned media (TCM) from LC3-547 cells treated with DMSO, MRTX, or Soto for subsequent analyses. All experiments were repeated at least 3 times, each time with 3 technical replicas. (E) qRT-PCR for Tgfa and Cxcl5 of LC3-547 cells treated with DMSO or 500 nM MRTX for 48 hours. Data are represented as mean ± SEM, and statistical significance was determined with a 2-tailed Student’s t test for unpaired samples. (F) Heatmap with z score of Luminex data of TCM from LC3-547 cells treated with 500 nM MRTX, 500 nM Soto, or equimolar concentration DMSO for 48 hours depicting cytokine and growth factor secretion. (G) Quantification (pg/mL) of indicated cytokines in TCM from treated LC3-547 cells. Data are represented as mean ± SEM. Statistical significance was determined using 1-way ANOVA with Tukey’s post hoc test. (H) Representative images of p-EGFR/ECAD/DAPI. Scale bars: 25 mM. (I) Quantification of percentage p-EGFR+ area of total ECAD+ area. (J) Representative Western blot of KRASG12D expression and p-ERK1/2 in human KRASG12D lung adenocarcinoma cell line, A427, upon 3 hours treatment with 1 μM MRTX or 1 μM Soto or equimolar DMSO. (K) Experimental design: RNA and TCM were harvested from human KRASG12D cancer cells cultured with DMSO, 100 nM MRTX, or 500 nM Soto. All experiments were repeated at least 3 times, each time with 3 technical replicas. (L) Quantification of CXCL1, CCL5, and TGF-α cytokines in A427 TCM from cells treated with DMSO, MRTX, or Soto. Data are represented as mean ± SEM and differences were evaluated by 1-way ANOVA with post hoc Tukey’s HSD test.