Immunological signatures from irradiated cancer-associated fibroblasts

Abstract

Introduction: Cancer-associated fibroblasts (CAFs) are abundant and influential elements of the tumor microenvironment (TME), giving support to tumor development in multiple ways. Among other mechanisms, CAFs are important regulators of immunological processes occurring in tumors. However, CAF-mediated tumor immunomodulation in the context of radiotherapy remains poorly understood. In this study, we explore effects of radiation on CAF-derived immunoregulatory signals to the TME.

Methods: Primary CAF cultures were established from freshly collected human NSCLC lung tumors. CAFs were exposed to single-high or fractionated radiation regimens (1x18Gy or 3x6Gy), and the expression of different immunoregulatory cell-associated and secreted signaling molecules was analyzed 48h and 6 days after initiation of treatment. Analyses included quantitative measurements of released damage-associated molecular patterns (DAMPs), interferon (IFN) type I responses, expression of immune regulatory receptors, and secretion of soluble cytokines, chemokines, and growth factors. CAFs are able to survive ablative radiation regimens, however they enter into a stage of premature cell senescence.

Results: Our data show that CAFs avoid apoptosis and do not contribute by release of DAMPs or IFN-I secretion to radiation-mediated tumor immunoregulation. Furthermore, the secretion of relevant immunoregulatory cytokines and growth factors including TGF-β, IL-6, IL-10, TNFα, IL-1β, VEGF, CXCL12, and CXCL10 remain comparable between non-irradiated and radiation-induced senescent CAFs. Importantly, radiation exposure modifies the cell surface expression of some key immunoregulatory receptors, including upregulation of CD73 and CD276.

Discussion: Our data suggest that CAFs do not participate in the release of danger signals or IFN-I secretion following radiotherapy. The immune phenotype of CAFs and radiation-induced senescent CAFs is similar, however, the observed elevation of some cell surface immunological receptors on irradiated CAFs could contribute to the establishment of an enhanced immunosuppressive TME after radiotherapy.

Keywords: CAFs; NSCLC; cancer-associated fibroblasts; immunosuppression; ionizing radiation; non-small cell lung cancer; radiotherapy; tumor microenvironment.

Figure 1

Effects of IR on CAF viability. Induction of cell senescence and apoptosis. Primary Human lung CAF cultures were checked for apoptosis (TUNEL assay—panel A) and senescence (β-Galactosidase assay – panel B) on day five after first radiation exposure. (A) Cell survival and apoptosis were measured by the TUNEL assay and assessed flow cytometry. Positive controls included human lymphoma cells treated with Camptothecin or CAFs treated with the pro-apoptotic agent Staurosporine. (B) Premature cell senescence (blue-stained cells) was induced by fractionated-medium and high-dose radiation regimens. Scale Bar = 15 μm. ****Statistically significant, with p<0.001.

Figure 2

CAF irradiation and generation of DAMPs. (A) Levels of extracellular ATP in irradiated Human lung CAF cultures were measured 24h post-radiation treatment. Cells treated with triton X-100 for 1h were used as positive control. Bars represent mean value (± SD) from two different CAF donors; (B) Expression of HMGB1 in Human lung CAF cell lysates and supernatants was analyzed by Western blots at 48h and 5 days post-radiation treatment; (C) Cell surface calreticulin expression on irradiated Human lung CAFs measured by flow cytometry. Bar graphs represent mean (± SD) values from flow cytometry analysis of 3 CAF donors, measured independently. (D) Micronuclei formation in irradiated Human lung CAFs measured by DAPI nuclear staining. (E) Bar graphs represent percentage of micronuclei positive cells (± SD) analyzed from 4 CAF donors, measured independently. (F) Effects of radiation on secretion of IFN-β by Human lung CAFs and A549 cells were analyzed by ELISA assay. IFN-β secretion in supernatants was determined 48h post-irradiation. Welch ANOVA test and p-values were determined between control and irradiated CAFs. *: Statistically significant, with p<0.05. **: Statistically significant, with p<0.01. ns: Not significant.

Figure 3

Radiation-induced effects on STAT-1 and NF-κB pathways in CAFs. (A) Western blot analysis, using anti-STAT1, p-STAT1 (T701), NF-κB/p65, and p-NF-κB/p65 (S536) on irradiated and non-irradiated Human lung CAF whole cell lysates were analyzed 5 days after irradiation. Results were normalized against β-actin expression. (B) Relative intensity of the bands corresponding to panel, determined by densitometry, is shown as a bar graph. Data represent mean (± SD) values from 5 different CAF donors. Two-way ANOVA test and p-values were determined individually between non-irradiated CAFs and the two irradiated CAF groups.

Figure 4

Secretion of inflammatory and immunoregulatory factors by irradiated Human lung CAFs. (A) The release of eleven subjectively selected inflammatory/immunoregulatory factors from CAFs was measured in CAF-CMs by ELISA and multiplex protein arrays, 5 days after radiation treatment. Mean values from five different CAF donor samples are shown. (B) Levels of active IFNs and TGF-β in CAF-CM were examined by checking induction of STAT-1 and Smad2/3 phosphorylation in A549 cells upon exposure to CAF-CM. Cultured A549 cells were exposed for 3h to CAF-CM, before cell lysis. Figure shows outcomes from 3 unrelated CAF donors.

Figure 5

Surface expression of immunoregulatory receptors on irradiated Human lung CAFs. (A) Gating strategy used to analyze expression of immunoregulatory receptors on CAFs, 5 days after radiation treatment. (B) Bar graphs represent mean (± SD) values from flow cytometry analysis of 5 randomly selected CAF donors, measured independently. Results are expressed as median fluorescence intensity (MFI). Welch ANOVA test and p-values were determined between control and irradiated CAFs individually. *: Statistically significant, with p<0.05. ns: Not significant.

Figure 6

Radiation decreased tumor volume and induced expression of immunoregulatory receptors in vivo. Subcutaneous (A) LLC tumors from (1x12Gy) irradiated and non-irradiated (0Gy) Black6 mice (n=4 per group) were quantified as a percentage of total tumor area. (B) Immunohistochemistry was performed to assess the expression of CD73 and CD276 in stromal cells from these tumors. Results represent the mean (± SD) values from 4 different tumors per group. IR, irradiation. Graphs represent optical densities of 3,3′-Diaminobenzidine (DAB) staining of 10 different fields from 4 different mice per group. **:Statistically significant, with p<0.01. ***: Statistically significant, with p<0.001.

Figure 7

Schematic overview of findings: Radiation-mediated effects on immunological signals in CAFs. Radiation promotes the acquisition of a senescence phenotype in CAFs with a concurrent reduction in cell proliferation and migration rates. Exposure of CAFs to ablative radiation doses is insufficient to induce release of DAMPs. Likewise, radiation is unable to trigger measurable IFN-I responses in CAFs. CAFs are engaged in the release of soluble inflammatory and immunoregulatory factors resembling the senescence-associated secretory phenotype (SASP), and this phenotype is mostly unchanged after radiation exposure. Surface expression of some important immunoregulatory receptors in CAFs becomes increased after radiation exposure.