Exosomes released by breast cancer cells under mild hyperthermic stress possess immunogenic potential and modulate polarization in vitro in macrophages

Abstract

Macrophages play a dual role in tumor initiation and progression, with both tumor-promoting and tumor-suppressive effects; hence, it is essential to understand the distinct responses of macrophages to tumor progression and therapy. Mild hyperthermia has gained importance as a therapeutic regimen against cancer due to its immunogenic nature, efficacy, and potential synergy with other therapies, yet the response of macrophages to molecular signals from hyperthermic cancer cells has not yet been clearly defined. Due to limited response rate of breast cancer to conventional therapeutics the development, and understanding of alternative therapies like hyperthermia is pertinent. In order to determine conditions corresponding to mild thermal dose, cytotoxicity of different hyperthermic temperatures and treatment durations were tested in normal murine macrophages and breast cancer cell lines. Examination of exosome release in hyperthermia-treated cancer cells revealed enhanced efflux and a larger size of exosomes released under hyperthermic stress. Exposure of naïve murine macrophages to exosomes released from 4T1 and EMT-6 cells posthyperthermia treatment, led to an increased expression of specific macrophage activation markers. Further, exosomes released by hyperthermia-treated cancer cells had increased content of heat shock protein 70 (Hsp70). Together, these results suggest a potential immunogenic role for exosomes released from cancer cells treated with mild hyperthermia.

Keywords: Hyperthermia; breast cancer; exosome; immune system; macrophages.

Figure 1.

(a, b) Cell viability of 4T1 and EMT-6 cells after treatment at 43° C determined using MTT assay. The cell viability of untreated cells was considered as 100%. (c, d) Cell viability of 4T1 and EMT-6 using Annexin V-FITC-PI-based FACS at CEM43 TD₅₀ [Individual replicate data provided in supplementary materials Figure S-3].

Figure 2:

NTA analysis of isolates of 4T1 and EMT-6 derived exosomes. (a, c) Data represents the number of exosomes obtained from isolation replicates (n = 3, p=0.0376 for 4T1, p=0.0174 for EMT-6). The exosome yield reported here has been standardized to exosomes per 10⁷ viable cells at the time of exosome isolation. (b, d) Data represents the average size distribution profile from isolation replicates (n = 3). Student's t-test was used for comparing data between the two groups. [Individual replicate data provided in supplementary materials Figure S-4, S-5].

Figure 3.

TEM images of negatively stained exosomes. (a) 4T1 control exosomes, (b) 4T1 exosomes post-hyperthermia treatment, (c) EMT-6 control exosomes, and (d) EMT-6 exosomes post-hyperthermia treatment. Yellow arrows signify the presence of relatively larger exosomes observed in greater abundance in hyperthermia-based treated sets.

Figure 4.

(a) Bright-field images of transwell migration of RAW 264.7 cells upon treatment with LPS (positive control), EMT-6 control exosomes, EMT-6 exosomes post-hyperthermia treatment, 4T1 control exosomes, and 4T1 exosomes post-hyperthermia treatment. (b) Transwell migratory profile of RAW 264.7 macrophages upon treatment with LPS (positive control), EMT-6 control exosomes, EMT-6 exosomes post-hyperthermia treatment, 4T1 control exosomes, and 4T1 exosomes post-hyperthermia treatment. Each bar represents the mean ± standard error (n=3). One-way ANOVA followed by Tukey's test was used for comparing data between groups, where *** indicates p <0.001 between bracketed groups.

Figure 5.

(i) SEM images of (a) Untreated negative control, (b) Positive control (LPS-treated), (c, e) control exosome-treated and (d, f) hyperthermia derived exosome treated RAW 264.7 cells (scale bar represents 10 μm), (ii) Percentage of flattened cells in positive control, post-hyperthermia derived exosome treated RAW 264.7 cells compared to respective controls after 24 hours treatment. Each bar represents the mean ± standard deviation with sample size (number of counted cells) >50. The student's t-test was used for comparing data between the two groups (n=3), where * indicates p <0.05.

Figure 6

Western blot analysis of Hsp70, CD9, CD63 proteins in exosomes from control untreated or hyperthermia treated EMT-6 and 4T1 cells with individual densitometry analysis. Each bar represents the mean optical density ± standard error (n=3). The student’s t-test was used for comparing data between the two groups (n=3), where * indicates p <0.05. The green arrow in (a) represents increased expression (signal intensity).

Figure 7.

Untreated or hyperthermia based exosome-mediated alterations in inflammatory or downstream signaling pathways as observed in RAW 264.7 macrophages. Each bar represents the mean concentration ± standard error (n=3). One-way ANOVA followed by Tukey's test was used for comparing data between groups, where * indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001 between bracketed groups.

Figure 8.

qRT-PCR profile of macrophage polarization marker expression (normalized to GAPDH) in RAW 264.7 after 24 hours exposure to control and hyperthermia based exosomes from 4T1 and EMT-6 cells, respectively. One-way ANOVA followed by Tukey's test was used for comparing data between groups, where * indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001 between bracketed groups (n=3).

Figure 9.

qRT-PCR profile of macrophage polarization marker expression (normalized to GAPDH) in BMDMs following the exposure to control exosomes or exosomes derived from 4T1 and EMT-6 cells exposed to hyperthermia. (a) Arg-1 profile of BMDM cells following 24 hours exposure to control and hyperthermia based exosomes from 4T1 and EMT-6 cells respectively, (b-c) iNOS profile of BMDM cells after 24 hours exposure to control exosomes or exosomes derived from 4T1 and EMT-6 cells exposed to hyperthermia. The Arg-1 and iNOS expression level in untreated control and EMT-6 control exosomes treated BMDM cells in (a,b) is close to zero and hence not visible in the graph. Similarly iNOS expression level is negligible in untreated control and 4T1 control exosome treated BMDM cells in (c) and hence not visible in the graph. One-way ANOVA followed by Tukey's test was used for comparing data between groups, where * indicates p <0.05, ** indicates p <0.01 and *** indicates p <0.001 between bracketed groups (n=3).