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Research ArticleOncology
Open Access | 
10.1172/jci.insight.182228
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Lasse-Opsahl, E. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Barravecchia, I. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by McLintock, E. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Lee, J. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Ferris, S. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Espinoza, C. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Hinshaw, R. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Cavanaugh, S. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Robotti, M. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Rober, L. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Brown, K. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Abdelmalak, K. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Galban, C. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
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1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Zhang, Y. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Pasca di Magliano, M. in: JCI | PubMed | Google Scholar
1Graduate Program in Cancer Biology,
2Department of Radiology, and
3Center for Molecular Imaging, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
4The Institute of Biorobotics, Scuola Superiore Sant’Anna, Pisa, Italy.
5Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
6PhD School in Translational Medicine, Scuola Superiore Sant’Anna, Pisa, Italy.
7Department of Biomedical Engineering,
8Rogel Cancer Center, and
9Department of Cell and Developmental Biology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Address correspondence to: Stefanie Galban, Center for Molecular Imaging, Department of Radiology, Department of Surgery, BSRB A502, The University of Michigan, Ann Arbor, Michigan 48109, USA. Phone: 734.764.4076; Email: sgalban@umich.edu. Or to: Marina Pasca di Magliano, Department of Surgery, Department of Cell and Developmental Biology, Rogel Cancer Center Room 6306, 1500 E. Medical Center Drive, The University of Michigan, Ann Arbor, Michigan 48109, USA. Email: marinapa@umich.edu.
Authorship note: ELLO and IB are co–first authors.
Find articles by Galban, S. in: JCI | PubMed | Google Scholar
Authorship note: ELLO and IB are co–first authors.
Published January 9, 2025 - More info
Published in Volume 10, Issue 1 on January 9, 2025
AbstractLung 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.
IntroductionIn 2024, an estimated 234,580 new patients will be diagnosed with lung cancer in the United States, and 80% of those will have non–small cell lung cancer (NSCLC) (1). NSCLC is subclassified into 3 major groups: adenocarcinomas (40%), squamous cell carcinomas (30%), and large cell carcinomas (15%). KRAS is commonly mutated in adenocarcinomas. The most common point mutations are G12C (39%), followed by G12V (21%), G12D (17%), and G12A (10%) (2). Notably, while KRASG12C is the most prevalent mutation in adenocarcinoma among former or current smokers (42%), KRASG12D is most common in patients who have never smoked (56%) (2). Nonsmokers have poorer prognosis with worse outcomes to immunotherapies than smokers (3), possibly due to lower mutation burden — and thus, lower neoantigen prevalence and fewer changes in the tumor microenvironment.
While mutant KRAS was long deemed undruggable, several isoform-specific inhibitors have recently entered the clinic. The KRASG12C inhibitors sotorasib and adagrasib have gained FDA approval for lung cancer, while MRTX1133, a KRASG12D inhibitor, has entered phase I/II clinical testing for malignancies, including G12D-mutant lung cancer. While these are exciting developments, the prospect of resistance to targeted therapy is real; resistance already has been described for G12C inhibitors (4), with some instances involving sotorasib linked to the remodeling of the tumor microenvironment (5, 6). The effects of KRASG12D inhibition in lung adenocarcinoma, including changes in the tumor microenvironment and mechanisms of resistance, remain largely unknown.
Genetically engineered mouse models (GEMMs) expressing oncogenic KRAS upon Cre recombination, with or without mutation of the tumor suppressor p53, model the development of NSCLC resembling the human disease and are widely used for both basic discovery and preclinical studies (7). Studies using a similar model that allows both inducible and reversible expression of oncogenic KRASG12D have revealed that this oncogene is required not only for tumor initiation, but also for tumor maintenance, independently of the presence of additional mutations (such as inactivation of Trp53 or other tumor suppressor genes) (8). However, the effect of oncogenic KRAS expression and subsequent inactivation in the tumor microenvironment in the lung remains unexplored. Given the likely need to devise combination targeting modalities to prevent or overcome resistance to targeted KRAS inhibitors, it is imperative to unravel the cellular crosstalk between KRAS-mutant epithelial cells and their surrounding microenvironment.
To study the extrinsic role of oncogenic KRAS in lung cancer maintenance and progression, we modified a GEMM that expresses mutant KRASG12D in an inducible and reversible manner in the lung epithelium. In this Lung-iKRAS (L-iKRAS) model, first described by the Varmus laboratory (8), expression of oncogenic KRAS in club cells, in combination with loss-of-function mutations in tumor suppressor genes, efficiently drives formation of lung adenocarcinoma in both male and female mice. Inactivation of oncogenic KRASG12D in tumors results in tumor regression, establishing a role for oncogenic KRASG12D in the maintenance of NSCLC.
We have re-derived the L-iKRAS model, incorporating current understanding of lung cancer etiology (9–11). While the initial model used a Trp53-null allele or Ink4a-null allele (8), we used an allele expressing mutant Trp53R172H — as mutant alleles are more common than null ones in human cancer — in an inducible manner (12), wherein mutant-p53 expression in the lung is activated by intranasal administration of adenovirus expressing Cre recombinase (ad-Cre) (8, 13). Here, we used the L-iKRAS model to drive NSCLC in mouse lungs; we then inactivated oncogenic KRAS and, as expected, observed tumor regression. Interestingly, characteristics of the microenvironment, such as fibroblast activation status, drastically changed upon KRAS inactivation. To gather an unbiased understanding of the effects of inactivating oncogenic KRAS, we performed single-cell RNA sequencing (scRNA-seq) on lungs of iKRAS mice with KRASG12D on (L-iKRASG12D ON) or lungs where KRASG12D had been inactivated (L-iKRASG12D OFF). In the presence of oncogenic KRAS, macrophages in the lung expressed immunosuppressive markers, consistent with the poor immunogenicity of KRASG12D-mutant tumors. To understand how oncogenic KRAS regulates the composition and differentiation status of macrophages, we used ex vivo approaches. Mechanistically, we show that fibroblasts are a key signaling hub, mediating interactions between epithelial cells and macrophages, and thus, potentially mediating the immunosuppressive status of tumors.
ResultsInhibition of KRASG12D results in tumor regression in L-iKRAS mouse model of lung adenocarcinoma. To generate a mouse model wherein oncogenic KRAS can be induced and reversed in the lung with accompanying mutant-p53 expression, we crossed the bitransgenic Ccsp-rtTa; TetO-KrasG12D mouse (8) with the Trp53LSL-R172H mouse (8, 14). In this study, we refer to this GEMM as L-iKRAS (Ccsp-rtTa; TetO-KrasG12D; Trp53LSL-R172H/+). Administration of doxycycline (dox) in drinking water induces expression of the reverse tetracycline transactivator (rtTa) in club cells of the lung, where it binds to the response element (TetO-KrasG12D) to initiate lung-specific expression of KRASG12D. When dox is withdrawn from drinking water, rtTa is inactivated and expression of KRASG12D is prevented/reversed in club cells. Conditional, lung-specific expression of mutant p53R172H in the lung can be achieved simultaneously by intranasal administration of ad-Cre at study start (Figure 1A). Single-transgenic mice given dox at the same concentration and for the same duration as experimental mice, or triple-transgenic mice that did not receive dox, served as controls for this study. Controls also included single- or triple-transgenic mice given ad-Cre. We compared overall survival of the 3 experimental cohorts to understand how KRASG12D expression alone, or KRASG12D with mutant p53 co-occurring mutation, impacts tumor growth in the lung (Figure 1B). The first experimental group (Ccsp-rtTa;TetO-KrasG12D with or without Trp53LSL-R172H/+) was given dox, but not ad-Cre, to induce expression of KRASG12D but not mutant p53. The control group, which included single-transgenic mice or Trp53LSL-R172H/+ mice lacking a Ccsp-rtTa or TetO-KrasG12D cassette, was given dox and ad-Cre for a period of 40 weeks. The second experimental group, a triple-transgenic cohort (Ccsp-rtTa; TetO-KrasG12D; Trp53LSL-R172H/+), was given dox and ad-Cre at study start to induce KRASG12D and mutant p53 expression. Mice expressing only KRASG12D had a median survival of 33 weeks, whereas mice expressing both oncogenic KRAS and mutant p53 had a median survival of only 17 weeks, with survival decreasing after week 10. Differences in overall survival were statistically significant between all groups. The decreased survival of the mutant KRAS and mutant p53 groups may indicate that disease severity increases when p53 is co-mutated, as well as a transition from adenomas to adenocarcinomas (Figure 1B). To investigate the effect of KRASG12D inhibition on tumor growth, L-iKRAS ON mice and control littermates (Control) were given ad-Cre and dox water for 17–25 weeks. Fisher et al. showed that at 17–25 weeks, mice develop a measurable tumor burden, thus providing rationale for our analyses (8). A second group, L-iKRAS OFF mice, was given ad-Cre for 17 weeks before dox withdrawal for 1, 2, or 4 weeks (Figure 1C). We assessed lung sections from each group for KRASG12D expression by Western blotting using a RASG12D-specific antibody. As expected, we detected RASG12D expression only in lung tissue from the ON group (Figure 1D). Next, we harvested lungs from all experimental cohorts to assess pulmonary lesions on hematoxylin and eosin–stained (H&E-stained) lung sections (Figure 1E). The percentage tumor area per lung area and the total number of lesions per lobe increased in the ON compared with the Control group and decreased statistically significantly when KRASG12D expression was reversed by dox removal for 1, 2, or 4 weeks (Figure 1F and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.182228DS1, respectively). Higher magnification images of H&E staining are presented in Supplemental Figure 1B. Furthermore, depletion of oncogenic KRAS decreased cell proliferation in E-cadherin+ (ECAD+) epithelial cells, as assessed by Ki67 staining in all groups (Figure 1G, higher magnification in Supplemental Figure 1C; quantified in Figure 1H). In lung sections stained with an anti–p-ERK1/2 antibody, p-ERK1/2 expression increased in ECAD+ epithelial cells in the ON group and decreased in the OFF groups (Figure 1I, higher magnification in Supplemental Figure 1D; quantified in Figure 1J). Pulmonary lesions disappeared in the OFF groups, likely because of apoptotic cell death described by Fisher et al. (8) and assessed here by cleaved caspase 3 (CC3) staining in ECAD+ epithelial cells (Figure 1K, higher magnification in Supplemental Figure 1E; quantified in Figure 1L).
Figure 1Inducible and reversible L-iKRASG12D mouse model of lung adenocarcinoma. (A) Schematic depicting L-iKRASG12D mouse model and its inducibility and reversibility of KRASG12D expression in club cells of the lung with dox and activation of mutant Trp53 expression by ad-Cre. (B) Kaplan-Meier survival analysis comparing control mice (n = 20, Ccsp-rtTa or TetO-KrasG12D with or without Trp53–/+ allele and with and without dox or ad-Cre), KrasG12D (n = 11, Ccsp-rtTa; TetO-KrasG12D; Trp53–/+ on dox, but no ad-Cre or Ccsp-rtTa; TetO-KrasG12D on dox, with or without ad-Cre), and KrasG12D/Trp53R172H/+ (n = 23, Ccsp-rtTa; TetO-KrasG12D; Trp53LSL-R172H/+ on dox plus ad-Cre). Log-rank (Mantel-Cox) test with statistically significant P value of 0.0033. Median survival for all groups is indicated in inset. One-way ANOVA with Tukey’s post hoc test showed the median survival of the KrasG12D/Trp53R172H/+ group was significantly lower than that of the control group (P value: 0.0014) and the KrasG12D group (P value: 0.0148). (C) Timeline for KRASG12D induction (ON) and KRASG12D inhibition (OFF) in triple-transgenic and control mice (single transgenics). (D) Western blot using anti-KRASG12D antibody or anti–total RAS (T-RAS) with corresponding β-actin blots as loading control of L-iKRAS lung tissue from all groups (ON: 20 weeks, OFF: 20 weeks ON and 4 weeks OFF). (E) Representative images of H&E. Scale bars: 50 mM. ON: 17–20 weeks, OFF 1 and 4 weeks. (F) Quantification of percentage tumor area over total lung area from whole slide scanned images in control, ON (17–25 weeks) and OFF (1, 2, and 4 weeks combined). (G) Representative images of Ki67/ECAD/DAPI. Scale bars: 25 mM. (H) Quantification of percentage Ki67+ cells among total ECAD+ cells. (I) Representative images of p-ERK/ECAD/DAPI. Scale bars: 25 mM. (J) Quantification of percentage p-ERK+ cells among total ECAD+ cells. (K) Representative images of CC3/ECAD/DAPI. Scale bars: 25 mM. (L) Quantification of percentage CC3+ cells among total ECAD+ cells. Data in F, J, H, and L are presented as mean ± SEM.
We also assessed inhibition by MRTX1133 (hereafter denoted MRTX), a clinically relevant KRASG12D inhibitor, as MRTX and other KRASG12D inhibitors are now in clinical trials. We orthotopically implanted KRASG12D-mutant KPL-86 cells derived from the KP model into the lungs of mice, and then treated mice with MRTX for 2 days at 30 mg/kg twice daily by i.p. injection (see schematic in Supplemental Figure 1F). Lungs were harvested and stained with H&E, Ki67, p-ERK1/2, and CC3 (Supplemental Figure 1, G–I). Ki67 and p-ERK1/2 staining indicated that MRTX treatment resulted in a decrease in proliferation and MAPK signaling. Furthermore, apoptotic cell death increased in MRTX-treated lung sections compared with vehicle-treated sections. In summary, we demonstrate that induction of KRASG12D and co-occurring mutant p53 expression in the lung produces highly proliferative lung tumors that can be reduced by abrogating KRASG12D expression, genetically or chemically, resulting in fewer pulmonary lesions, likely due to apoptotic cell death of tumor cells.
Oncogenic KRAS results in gene expression changes in epithelial cells in the tumor. To better understand KRASG12D-dependent changes occurring in the tumor and tumor microenvironment, we utilized scRNA-seq. We harvested the right inferior lobe of lungs from male and female mice in the ON (21 weeks) and OFF (21 weeks ON, 1 week OFF) groups. Samples were processed simultaneously to avoid batch effects. The processed scRNA-seq dataset had 14,005 cells: 9,090 from the ON group and 4,915 from the OFF group. Data were analyzed using the Seurat package in R (version 4.0.2) and visualized by uniform and manifold approximation and projection (UMAP) (Figure 2A). We observed no differences between male and female mice. As shown in Figure 2B, clusters from the ON and OFF groups overlaid sufficiently, indicating that similar cell types were present in both groups. Of note, we observed a higher percentage of fibroblasts in the ON compared with the OFF group and a higher percentage of macrophages in the OFF compared with the ON group (Figure 2C). To parse differences between groups in lung epithelia, we subclustered epithelial cells separately and visualized clusters by UMAP (Figure 2D). We assessed changes in gene expression in the epithelia; interestingly, genes upregulated in the OFF group included chemokines Cxcl2 and Ccl4; Wnt4, a noncanonical WNT signaling ligand; growth factor Fgf1; and the extracellular matrix remodeling factor Timp3. Genes upregulated in the ON group included Vegfa and Cxcl15 (Figure 2, E and F, and Supplemental Figure 2A). We also assessed expression changes for genes that change during lung cancer progression, including Cxcl1, Tgfa, and Tgfb1. While Cxcl1 expression was upregulated in the OFF group, Tgfa expression was higher in the ON group; Tgfb1 expression showed no change (Supplemental Figure 2B). Using gene set enrichment analysis (GSEA), we confirmed that KRAS signaling was efficiently abrogated in the OFF group, as the “HALLMARK_KRAS_SIGNALING_DN” gene set, a list of genes that are downregulated upon KRAS expression, was significantly upregulated in the OFF group (Figure 2G). Conversely, the “HALLMARK_KRAS_SIGNALING_UP” gene set, a list of genes that are upregulated upon KRAS expression, trended up in the ON group (Supplemental Figure 2C).
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