SHP-2 and PD-L1 Inhibition Combined with Radiotherapy Enhances Systemic Antitumor Effects in an Anti-PD-1-Resistant Model of Non-Small Cell Lung Cancer

Abstract

Immune checkpoint inhibitors, such as anti-PD-1/PD-L1, have emerged as promising therapies for advanced non-small cell lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD-1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with anti-PD-L1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti-PD-1-resistant mouse model. We treated 129Sv/Ev mice with anti-PD-1-resistant 344SQ NSCLC adenocarcinoma with oral SHP099 (a SHP-2 inhibitor) combined with XRT and intraperitoneal anti-PD-L1. Primary tumors were treated with XRT (three fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. XRT in combination with SHP099 and anti-PD-L1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. XRT also increased SHP-2⁺ M1 tumor-associated macrophages in abscopal tumors (P = 0.019). The addition of SHP099 also associated with a higher M1/M2 ratio, greater numbers of CD8⁺ T cells, and fewer regulatory T cells. This triple-combination therapy had strong antitumor effects in a mouse model of anti-PD-1-resistant NSCLC and may be a novel therapeutic approach for anti-PD-1-resistant NSCLC in patients.

Figure 1.. Triple therapy with radiation+SHP099+anti–PD-L1 has antitumor effects in the primary and abscopal tumors, extends survival, and suppresses lung metastases.

(A) The indicated numbers of 344SQR non–small-cell lung cancer (NSCLC) tumor cells were inoculated subcutaneously into the right hind leg (primary tumor; irradiated) and left hind leg (secondary or abscopal [unirradiated] tumor) of mice, which were then treated at the indicated times with radiotherapy (XRT; three 12-Gy fractions), SHP099, anti–PD-L1, anti–PD-L1+SHP099, XRT+anti–PD-L1, or XRT+anti–PD-L1+SHP099 (n=8 mice/group). (B-C) Growth curves for tumors at primary (B, left axis) and secondary (C, right axis) sites. Data are presented as mean±SEM, with P values derived from two-way analysis of variance. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; n.s., not significant. (D) Survival curves for the different treatment groups. P values were calculated with log-rank tests. ***P<0.001; ****P<0.0001. (E) Mice in the different treatment groups were euthanized and lungs were harvested for metastases counts after staining with Bouin’s fixative solution. The lung metastasis ratio was defined as the ratio between lung metastasis counts and survival days. Data are presented as mean+SD, with P values derived from t tests. *P<0.05; **P<0.01; ***P<0.001. (F) Representative photographs of primary tumors (right leg, green arrow), abscopal tumors (left leg, red arrow), and corresponding lung metastases at death in the control and triple-therapy groups.

Figure 2.. Nanostring analysis of the immune microenvironment in primary and abscopal tumors.

RNA from each tumor was isolated and subjected to comprehensive immune profiling with the NanoString nCounter PanCancer Mouse Immune Profiling platform. Genes associated with the indicated immune cells were analyzed and scored with the Advanced Analysis module of nSolver. (A-B) Heat maps for expression of genes associated with the indicated types of immune cells in primary (A) and abscopal (B) tumors in the various treatment groups (n=3 mice/group). (C-D) Z-scores of each pathway profiled in primary (C) and abscopal (D) tumors. (E-F) Specific pathway scores for the various treatment groups in primary (E) and abscopal (F) tumors. Data are presented as mean±SD, with P values derived from t tests. *P<0.05; **P<0.01

Figure 3.. Triple therapy increases the M1/M2 ratio.

A-F) Flow cytometric analysis of M41 and M2 tumor-associated macrophages (TAMs) on day 21 (11 days after radiotherapy; n=3 mice/group). Percentages of M1 (A) and M2 (BTAMs analyzed in primary tumors in the indicated treatment groups. M1 TAMs: CD11b⁺Gr1^intF4/80^highCD38^high; M2 TAMs: CD11b⁺Gr1^intF4/80^highCD206^high. (C) Representative flow cytometry panels for TAMs in primary tumors. M1 (D) and M2 (E) TAMs in abscopal tumor sites. (F) Representative flow cytometry panels for TAMs in abscopal tumors. (G-H) Nanostring molecular analysis was performed to confirm flow cytometry data for TAMs in irradiated and abscopal tumors. Top: Heatmaps for expression of genes related to M1 or M2 TAMs in primary (irradiated; G) and secondary (abscopal, unirradiated; H) tumors in the various treatment groups. Bottom: Fold-change in expression of the indicated genes for primary (G) and secondary (H) tumors. Data are presented as mean+SD and P values were derived from t tests. *P<0.05, **P<0.01, ***P<0.001; n.s., not significant. Flow data performed twice under the same schedule to confirm the results.

Figure 4.. Triple therapy increases CD8⁺ T cells and Tregs.

Flow cytometric analysis of lymphoid subtypes from tumors in the indicated treatment groups on day 21 (n=3 mice/group). (A) Frequency of CD8⁺ T cells in primary tumors. (B) Frequency of CD8⁺ T cells in abscopal tumors (C) Frequency of Tregs in primary tumors. (D) Frequency of Tregs in abscopal tumors. (E-F) Nanostring data (3 mice/group) for cytotoxicity- and activation-related genes. (E) Primary tumor expression of Gzma (P=0.001), Gzmb (P=0.016), and Ifng (P=0.031) vs. the control group. (F) Abscopal tumor expression of Gzmb (P=0.012), Gzmk (P=0.018), and Prf1 (P=0.02) compared with the control group. Data are presented as mean+SD, and P values were derived from t tests. *p<0.05, **P<0.01, ***P<0.001. Flow data performed twice under the same schedule to confirm the results.

Figure 5.. SHP-2 is expressed mainly in M1 tumor-associated macrophages (TAMs) and radiation (XRT) may further induce its expression.

On Day 21 (11 days after XRT), immune cells were isolated and phenotyped by flow cytometry from both primary and abscopal tumors (n=3 mice/group). (A−D) In primary tumors, SHP-2 was expressed mainly in TAMs, followed by tumor-associated neutrophils (TANs), Tregs, CD8⁺, and CD4⁺ T cells. (A) Graphic representation of SHP-2 mean fluorescence intensity (MFI) for the indicated cell types. (B) Quantification of SHP-2 MFI in the various immune cell subgroups (P<0.0001 for each comparison). (C) Representative plots for the percentages of SHP-2⁺ cells in the various immune cell subgroups. (D) Frequency of SHP-2⁺ cells for the subsets indicated (P<0.0001 for each comparison). (E−H) In primary tumors, SHP-2 MFI (E, F) and SHP-2⁺ populations (G,H) were had higher expression in M1 TAMs than M2 TAMs. (E) Graphic representation of SHP-2 MFI. (F) Quantification of SHP-2 MFI in M1 TAMs and M2 TAMs (P=0.0001). (G) Representative plots for the percentage of SHP-2⁺ M1 and M2 cells. (H) Frequency of SHP-2⁺ M1 TAMs and M2 TAMs (P=0.005). (I) Representative plots for SHP-2 expression after XRT. (J) SHP-2 expression after XRT in M1 TAMs from abscopal tumors (P=0.019) and primary tumors (P=0.084). Data are presented as mean+SD, with P values derived from t tests. *P<0.05, **P<0.01, ***P<0.001. Flow data performed twice under the same schedule to confirm the results.

Figure 6.. Depletion of CD8⁺ T cells and TAMs abolishes the effect of triple therapy.

(A) Mice (n=5/group) were inoculated with anti–PD-1–resistant 344SQR cells, as described in the text and in Figure 1A and given 1 of 5 treatments: No treatment (control), triple therapy (radiation [XRT]+anti–PD-L1+SHP099), triple therapy with CD8⁺ T-cell depletion, triple therapy with TAM depletion, and triple therapy with both TAM and CD8⁺ T-cell depletion. Mouse survival was recorded and compared among treatment groups with log-rank tests. (A) Survival in the non-depleted group, CD8⁺ T cell–depleted group (P=0.01), the F4/80⁺-depleted condition (P=0.0041), and the combined CD8⁺- and F4/80⁺-depleted condition (P=0.0004). P values were calculated with log-rank tests. *P<0.05; **P<0.01; ***P<0.001. (B) Relative to triple therapy, the lung metastasis ratio in the CD8⁺ depletion (P=0.007), F4/80⁺ depletion (P=0.003), and CD8⁺/ F4/80⁺ double-depletion (P=0.0006) groups. Data are presented as mean+SD, with P values derived from t tests. *P<0.05; ***P<0.001. (C-D) Effects of triple therapy with and without CD8⁺ or F4/80⁺ depletion on both primary and abscopal tumor growth.

Figure 7.. Schematic diagram.

Irradiated tumor (left panel): For the primary tumor, radiation increases M2 macrophages. SHP099, which repolarizes M2 to M1, and anti–PD-L1, synergistically boosts local control. Abscopal tumor (right panel) For the abscopal tumor, radiation increases M1 TAMs, including SHP-2⁺ M1 TAMs. Anti–PD-L1 and SHP099, which further polarize M2 to M1 TAMs, synergistically boosts the abscopal antitumor effect. Lightning bolt: radiation (XRT).