Tumor microenvironment modulation enhances immunologic benefit of chemoradiotherapy

Abstract

Background: Chemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects.

Methods: Therefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes.

Results: We show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8⁺ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8⁺ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner.

Conclusions: Overall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation.

Keywords: Chemoradiotherapy; Cyclophosphamide; Head and neck cancer; Head and neck squamous cell carcinoma; Human papillomavirus (HPV); Immunotherapy; Inducible nitric oxide synthase (iNOS); L-n6-(1-iminoethyl)-lysine (L-NIL); Radiotherapy; Tumor microenvironment.

Fig. 1

CTX / L-NIL reverses the unfavorable immune microenvironment of CRT treated tumors. a-c Subcutaneous established mEER tumors (day 17–18 post tumor cell injection) were treated with tumor-directed radiation (5 X 3Gy) and/or weekly cisplatin (83 μg/mouse) i.p. injections, according to the schedule in (a); mice were euthanized when tumors reached 225 mm². b Average tumor area (top) and statistical comparison of tumor sizes at time of first euthanasia (bottom; Tukey’s multiple comparison test). c Survival curves (top) and statistical comparison between treatments (bottom; Log-rank test); (b and c; N = 1 representative of 2; n = 6–8/group). d Heatmap depicting the relative abundance of various immune cell populations from CyTOF analysis in HPV16 immune reactive (IR+) and non-reactive (IR-) OPSCC human tumors based on the presence of HPV16-specific tumor infiltrating lymphocytes (TIL). Frequencies of DC (CD14-HLADR⁺CD11c⁺), M1 macrophages (CD163⁻CD14⁺HLA-DR⁺), monocytic MDCS (mMDSC; CD14⁺HLADR⁻), immature B cells (IgM⁺CD38⁺CD20⁺), memory B cells (IgM⁻CD38⁻CD20⁺), total CD4 and CD8⁺ T cells, naïve (Tn; CD45RA⁺CCR7⁺), central memory (Tcm; CD45RA⁻CCR7⁺) and effector memory (Tem; CD45RA⁻CCR7⁻) CD4⁺ and CD8⁺ T cells, CD4⁺CD161⁺ Tem, CD103⁺CD161⁻ and CD103⁺CD161⁺ CD8⁺ T cells. Th-denotation in indicates amount of IFNγ (Th1), IL-5 (Th2) and IL-17A (Th17) produced by the total TIL culture upon phytohemagglutinin stimulation. All data depicted as log-transformed values (base 2) relative to the median for each individual parameter and each column represents an individual patient (n = 9 total patients). e-f Established mEER tumors were treated with CRT and/or CTX/L-NIL immunomodulation (CTX at 2 mg/mouse i.p. and L-NIL at 0.2% in drinking water) and total tumor RNA was extracted and processed for gene expression analysis after 1 week of treatment, according to schedule in (e). f Heatmap of differential immune gene-set pathway enrichment represented as z-scores between treatment groups (See Additional file 1: Table S3 for immune pathway gene list). g Gene-set based immune cell type enrichment comparing CRT and CRT + CTX/L-NIL represented as z-scores (left; See Additional file 1: Table S4 for immune cell type gene list) and statistical comparison for each immune cell type (right; unpaired t test); (e and f; N = 1; n = 9/group). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant

Fig. 2

CTX/L-NIL combines with CRT to induce durable control of established tumors. Mice bearing established mEER tumors were treated with CRT (10 X 3Gy tumor-directed radiation and 83 μg/mouse weekly cisplatin i.p.) and/or CTX/L-NIL immunomodulation (CTX at 2 mg/mouse i.p. and L-NIL at 0.2% in drinking water) according to the schedule in (a); mice were euthanized when tumors reached 225 mm². b Individual tumor growth curves shown by treatment group, with each mouse represented as a single line. c Average tumor area (top) and statistical comparison at time of first euthanasia (bottom; Tukey’s multiple comparison test). d Survival curves (top) and statistical comparison between treatments (bottom; Log-rank test). (b-d; N = 1 representative of 2; n = 8–9/group). **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant

Fig. 3

CRT + CTX/L-NIL reprograms the myeloid tumor microenvironment. Established mEER tumors were treated with CRT and/or CTX/L-NIL and harvested after the first week of treatment, as shown in to Fig. 1e, and tumor myeloid cell infiltrate was analyzed using flow cytometry (a-e; see Additional file 1: Figure S4 for myeloid flow cytometry gating strategy) and ex vivo co-culture (f-h). a Myeloid-focused tSNE (among CD11b⁺/CD11c⁺ cells) from flow cytometry data for each treatment group. b Corresponding tSNE color-map (left) and radar plot (right) showing myeloid sub-type alterations between each treatment group as z-scores (myeloid sub-type color in radar plot corresponds with their color in tSNE map; N = 1 representative of 3; n = 8–9/group). c-e Percentage of myeloid sub-types among total myeloid cell tumor infiltrate (CD11b⁺/CD11c⁺), including inflammatory monocytes (c), M1-like macrophages (d), and granulocytic MDSCs (e) (N = 3; n = 23–25/group; Tukey’s multiple comparison test for inflammatory monocytes and Dunn’s multiple comparison test for M1-like macrophages and granulocytic MDSCs). f Experimental schematic used to test treatment-induced myeloid influence on CD8⁺ T cell cytotoxicity. Naïve splenic CD8⁺ T cells were stimulated with anti-CD3/CD28 antibodies and IL-2 in the absence or presence of myeloid cells (CD11b⁺ and CD11c⁺ cells) isolated from tumors that received different treatments for 3 days and then co-cocultured with mEER cells in presence of IL-2. After 24 h, apoptotic tumor cells were detected by Annexin V/ PI staining. g Representative images of mEER tumor cells stained for Annexin V (shown in green) following co-culture (scale bars show 200 μm). h mEER tumor cell apoptotic fold change (Annexin V⁺ / PI⁻) normalized to non-primed T cells (T cells not co-cultured with myeloid cells) (N = 4; n = 20–26/group; Dunn’s multiple comparison test). All bar graphs show mean +/− SD and each dot represents an individual mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

CRT + CTX/L-NIL promotes intratumoral CD8⁺ T cell infiltration and activation. Established mEER tumors were treated with CRT and/or CTX/L-NIL and harvested after the first week of treatment, as shown in Fig. 1e, and tumor lymphocyte infiltrate was analyzed using quantitative multiplex immunofluorescence (qmIF; a-c). a Representative multiplex images of mEER tumors showing DAPI (nuclei, dark blue), EpCAM (tumor, red), CD8α (CD8⁺ T cells, cyan), and Granzyme B (T cell cytotoxic marker, green). b Pie-charts showing T cell subset fractions among total T cells for each treatment (fraction averaged across 5 images per tumor and n = 3/group). c Log₂ fold change of Granzyme B (GzB)⁺ CD8⁺ cells normalized to control group (N = 1; n = 3/group; Tukey’s multiple comparison test). Bar graphs show mean +/− SD. *p < 0.05; **p < 0.01

Fig. 5

CRT + CTX/L-NIL enhances the lymphoid tumor microenvironment. Established mEER tumors treated with CRT and/or CTX/L-NIL and were harvested similar to Fig. 1e, and tumor lymphocyte infiltrate was assessed using flow cytometry (a-e; see Additional file 1: Figure S6 for lymphocyte flow cytometry gating strategy). a Lymphoid-focused tSNE (among TCRβ⁺ cells) from flow cytometry data for each group of treatment (left) and corresponding color-map (right) with colored lymphocyte subsets listed below (N = 1 representative of 5; n = 8–9/group). b Percentage of lymphoid sub-types among total tumor infiltrating T cells (TCRβ⁺ cells), including CD8⁺ T cells (top right), CD4⁺ T cells (bottom left) and regulatory T cells (bottom right) (N = 3–4; n = 23–36/group; Tukey’s multiple comparison test for CD8⁺ T cells and Dunn’s multiple comparison test for CD4⁺ and regulatory T cells). c Ratio of CD8⁺ T cells/ regulatory T cells percentages (N = 3; n = 23–24/group; Dunn’s multiple comparison test). d Percentage of E7-Tetramer⁺ among CD8⁺ T cells (N = 5; n = 33–37/group; Dunn’s multiple comparison test). e Representative flow cytometry histograms showing KLRG-1 expression among CD8⁺ T cells expressed as the % of the maximum count (left) and cumulative median fluorescence intensity (MFI) for each treatment group (right). FMO (Fluorescence minus one) is a mixture of all antibodies permitting CD8⁺ T cell identification without the phenotypical marker of interest (N = 2, n = 12; Tukey’s multiple comparison test). All bar graphs show mean +/− SD and each dot represents an individual mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 6

Tumor growth inhibition induced by CRT + CTX/L-NIL requires CD8⁺ T cells. Established mEER tumors were treated with CRT + CTX/L-NIL and anti-CD8α depleting antibody, or isotype control antibody, according to the schedule in (a); mice were euthanized when tumors reached 225 mm². b CD8⁺ T cell percentages among total viable cells in the blood at day 29 of treatment as assessed by flow cytometry (N = 1; n = 10/group, each as an individual dot; data are means +/− SD; Mann-Whitney test). c Individual tumor growth curves shown by treatment group, with each mouse represented as a single line. d Average tumor area (top) and statistical comparison at time of first euthanasia (bottom; Tukey’s multiple comparison test). e Survival curves (top) and statistical comparison between treatments (bottom; Log-rank test). (c-e; N = 1 representative of 2; n = 10/group). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant

Fig. 7

CTX/L-NIL immunomodulation renders the tumor microenvironment receptive to CRT. Schematic abstract: The tumor immune microenvironment (both myeloid and lymphoid compartments) remains “cold” after CRT treatment, and is characterized by the infiltration of granulocytic MDSCs and regulatory T cells. However, the combination of CRT with CTX/L-NIL reverses these immunosuppressive effects and further promotes increases in M1-like macrophages and inflammatory monocytes in the tumor. This favorable myeloid alteration likely plays a role in the improved intratumoral CD8⁺ T cell response, which shows enhanced specificity, effector and memory phenotypes