**3. Results and Discussion**

Several studies have shown that PDT is capable of triggering immune responses against tumor antigens [5,14,15]. One of the candidate mechanisms underlying this immune effect is the occurrence of ICD in cancer cells exposed to PDT [5,7]. Thus, the main goal of the present work was to verify whether two critical variables of PDT protocols, namely

photosensitizer concentration, and light dose, affect the ability of this approach to induce ICD in two different murine cancer cell lines, 4T1 and CT26, in vitro. CT26 and 4T1 cells were subjected to different PDT-AlPc-NE protocols to obtain IC50 and IC90. The concentrations obtained for CT26 can be found in the Supplementary Materials (Figures S1 and S2). The data for 4T1 cells were published in Rodrigues et al. [1].

ICD is characterized by cell death by apoptosis with a well-defined pattern of DAMP exposure. It is well described that PDT can preferentially trigger apoptosis or necrosis depending on protocol parameters such as the concentration of photosensitizer and the energy dose applied [1,7]. The results in Figure 1 corroborate findings in the literature, as the cell mechanism triggered by PDT depended on the protocol parameters. Both CT26 and 4T1 cells succumbed to apoptosis when submitted to PDT with their respective AlPc-NE IC50, 12.2 nM, and 9.01 nM, respectively, and the same energy dose—25 J/cm<sup>2</sup> . As expected, with the same AlPc-NE concentrations, but under a higher energy dose—67 J/cm<sup>2</sup> , a significant increase in the percentage of necrotic cells was observed. Moreover, necrosis was predominant in both CT26 and 4T1 cells exposed to their respective AlPc-NE IC90–31.5 and 19.4 nM–at both 25 J/cm<sup>2</sup> and 67 J/cm<sup>2</sup> energy doses (Figure 1). *Pharmaceutics* **2022**, *14*, 196 6 of 14

**Figure 1.** The induction of necrosis and apoptosis by PDT-AlPc-NE is affected by both the concentration of photosensitizer and the energy dose. (**A**) CT26 cells analyzed by the AO/PI method after 4 h of treatment; (**B**) CT26 cells analyzed by AnV/PI method after 24 h of treatments; (**C**) 4T1 cells analyzed by the AO/PI method after 4 h of treatment, and (**D**) 4T1 cells analyzed by the AnV/PI method after 24 h of treatments. Light gray bars represent the results of apoptotic cells, and dark gray bars represent necrotic cells. Untreated cells are represented with CT26 and 4T1.PDT protocols for CT26 cells: PDT1 = 12.2 nM and 25 J/cm²; PDT2 = 31.5 nM and 25 J/cm²; PDT3 = 12.2 nM and 67 J/cm²; and PDT4 = 31.5 nM and 67 J/cm².PDT protocols for 4T1 cells: PDT1 = 9.01 nM and 25 J/cm²; PDT2 = 19.4 nM and 25 J/cm²; PDT3 = 9.01 nM and 67 J/cm²; and PDT4 = 19.4 nM and 67 J/cm². MTX: mitoxantrone; F-T: three cycles of freeze-thawing; AO/PI: acridine orange and propidium iodide; AnV/PI: Annexin V and propidium iodide. Superscripts \* and \*\*\*\* represent *p* < 0.05 and *p* < 0.0001 relatives to apoptotic and necrotic cells of the same group. Data are presented as mean ± SEM for triplicates. **Figure 1.** The induction of necrosis and apoptosis by PDT-AlPc-NE is affected by both the concentration of photosensitizer and the energy dose. (**A**) CT26 cells analyzed by the AO/PI method after 4 h of treatment; (**B**) CT26 cells analyzed by AnV/PI method after 24 h of treatments; (**C**) 4T1 cells analyzed by the AO/PI method after 4 h of treatment, and (**D**) 4T1 cells analyzed by the AnV/PI method after 24 h of treatments. Light gray bars represent the results of apoptotic cells, and dark gray bars represent necrotic cells. Untreated cells are represented with CT26 and 4T1.PDT protocols for CT26 cells: PDT1 = 12.2 nM and 25 J/cm<sup>2</sup> ; PDT2 = 31.5 nM and 25 J/cm<sup>2</sup> ; PDT3 = 12.2 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 31.5 nM and 67 J/cm<sup>2</sup> . PDT protocols for 4T1 cells: PDT1 = 9.01 nM and 25 J/cm<sup>2</sup> ; PDT2 = 19.4 nM and 25 J/cm<sup>2</sup> ; PDT3 = 9.01 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 19.4 nM and 67 J/cm<sup>2</sup> . MTX: mitoxantrone; F-T: three cycles of freeze-thawing; AO/PI: acridine orange and propidium iodide; AnV/PI: Annexin V and propidium iodide. Superscripts \* and \*\*\*\* represent *p* < 0.05 and *p* < 0.0001 relatives to apoptotic and necrotic cells of the same group. Data are presented as mean ± SEM for triplicates.

The profile of DAMPs released by cells succumbing to PDT was also investigated. The exposure of CRT on the plasma membrane is an essential feature of ICD, as this DAMP facilitates the recognition and phagocytosis of the target cell by antigen-presenting cells [13,16,17]. These, in turn, will present the processed tumor cell antigens, potentially inducing an antitumor immune response mediated by CD8+ T cells [18,19]. Moreover, the exposure of HSP70 and HSP90 can increase the immunogenicity of the cells [20,21]. As shown in Figure 2, the exposure of HSP70, HSP90, and CRT were affected by variations in PDT-NE-AlFtCl parameters in both CT26 and 4T1 cells. The images used for fluores-It is noteworthy that the results profiles observed for both cell lines treated with IC<sup>50</sup> and 25 J/cm<sup>2</sup> were similar to those obtained with the ICD-positive control MTX. As expected, F-T induced necrosis in both cells studied. The treatments with MTX and F-T are often used as positive controls for apoptosis and necrosis, respectively [7,13]. Thus, variations in the PDT-AlPc-NE protocol parameters affect the type of cell death induced in the studied cells, which can be correlated to the intensity of the oxidative stress in the target cell following PDT.

cence quantification can be found in the Supplementary Materials (Figures S3–S5). When these cells were submitted to PDT with AlPc-NE IC50 and an energy dose of 25 J/cm2, a

centrations and higher energy doses. Other studies have also shown this same dose-dependency concerning DAMP exposure [22,23]. As expected, MTX induced HSP70, HSP90, and CRT exposure, while the F-T process did not cause the exposure of these DAMPs on the plasma membrane. The apparent increased exposure of HSP70 and HSP90 in CT26 cells treated with PDT4 is most probably due to the disruption of the plasma membrane that occurs in necrotic cells, which enables for the staining of these intracellular proteins

Another important hallmark of ICD is the release of HMGB1 to the extracellular medium [24–26]. HMGB1 is a nuclear protein associated with nucleosomes [26]. HMGB1 protein is released during the late phase of ICD, since long after the onset of apoptosis, and the chromatin becomes deconcentrated and, consequently, HMGB1 release occurs [24,26,27]. This DAMP attracts DC cells and macrophages; upon recognition, these cells

by immunofluorescence.

The profile of DAMPs released by cells succumbing to PDT was also investigated. The exposure of CRT on the plasma membrane is an essential feature of ICD, as this DAMP facilitates the recognition and phagocytosis of the target cell by antigen-presenting cells [13,16,17]. These, in turn, will present the processed tumor cell antigens, potentially inducing an antitumor immune response mediated by CD8<sup>+</sup> T cells [18,19]. Moreover, the exposure of HSP70 and HSP90 can increase the immunogenicity of the cells [20,21]. As shown in Figure 2, the exposure of HSP70, HSP90, and CRT were affected by variations in PDT-NE-AlFtCl parameters in both CT26 and 4T1 cells. The images used for fluorescence quantification can be found in the Supplementary Materials (Figures S3–S5). When these cells were submitted to PDT with AlPc-NE IC<sup>50</sup> and an energy dose of 25 J/cm<sup>2</sup> , a more intense CRT exposure, HSP70, and HSP90 was observed. A significantly lower exposure of these DAMPs was observed with the PDT protocols based on higher AlPc concentrations and higher energy doses. Other studies have also shown this same dose-dependency concerning DAMP exposure [22,23]. As expected, MTX induced HSP70, HSP90, and CRT exposure, while the F-T process did not cause the exposure of these DAMPs on the plasma membrane. The apparent increased exposure of HSP70 and HSP90 in CT26 cells treated with PDT4 is most probably due to the disruption of the plasma membrane that occurs in necrotic cells, which enables for the staining of these intracellular proteins by immunofluorescence.

Another important hallmark of ICD is the release of HMGB1 to the extracellular medium [24–26]. HMGB1 is a nuclear protein associated with nucleosomes [26]. HMGB1 protein is released during the late phase of ICD, since long after the onset of apoptosis, and the chromatin becomes deconcentrated and, consequently, HMGB1 release occurs [24,26,27]. This DAMP attracts DC cells and macrophages; upon recognition, these cells become mature and responsible for activating T cells [13,28]. The results presented in Figure 2 show that all the tested PDT protocols induced the release of HMGB1 by both CT26 and 4T1 cells, with a more intense release being observed with the protocol with AlPc IC<sup>50</sup> and energy dose of 25 J/cm<sup>2</sup> .

The release of the proinflammatory cytokine IL-1β was also assessed. For both the 4T1 and CT26 lines, only PDT with AlPc IC<sup>50</sup> and 25 J/cm<sup>2</sup> , and MTX, promoted a significant release of IL-1β compared to control (Figure 2G). IL-1β can modify the activity of many immune cell types, such as monocytes, macrophages, neutrophils, and lymphocytes, and can induce the release of other essential cytokines and chemokines involved in the activation of adaptive immune responses [29,30].

Thus, the in vitro experiments suggest that PDT-AlPc-NE can induce apoptosis and DAMPs exposure, a death pattern characteristic of ICD, in CT26 cells and 4T1 cells. Moreover, the results evidence that both the percentage of apoptotic cells and the DAMPs release profile are affected by the PDT parameters, specifically the AlPc-NE concentration and the energy dose.

As suggested by the literature [31], the immunogenicity of cells undergoing ICD can be assessed in in vivo vaccination-challenge models. Thus, CT26 cells and 4T1 cells subjected to different in vitro PDT protocols were used as prophylactic vaccines injected subcutaneously into the flank of the animals, as shown in Figure 3. The results show that 50% and 40% of mice vaccinated with CT26 cells treated with PDT1 [12.2 nM and 25 J/cm<sup>2</sup> ] and MTX, respectively, did not develop tumors up to 250 days after the challenge (Figure 3B). This result further shows that PDT can elicit ICD, an event already described in the literature. According to Garg et al. [21], 70% of mice vaccinated with CT26 cells treated with hypericin-mediated PDT were tumor-free.

and energy dose of 25 J/cm².

the activation of adaptive immune responses [29,30].

become mature and responsible for activating T cells [13,28]. The results presented in Figure 2 show that all the tested PDT protocols induced the release of HMGB1 by both CT26 and 4T1 cells, with a more intense release being observed with the protocol with AlPc IC50

The release of the proinflammatory cytokine IL-1β was also assessed. For both the 4T1 and CT26 lines, only PDT with AlPc IC50 and 25 J/cm², and MTX, promoted a significant release of IL-1β compared to control (Figure 2G). IL-1β can modify the activity of many immune cell types, such as monocytes, macrophages, neutrophils, and lymphocytes, and can induce the release of other essential cytokines and chemokines involved in

**Figure 2.** PDT-AlPc-NE induces the release of DAMPs by murine colorectal carcinoma (CT26) and murine mammary adenocarcinoma (4T1) cells. Surface CRT (**A**,**B**), surface HSP70 (**C**,**D**), and surface HSP90 (**E**,**F**). Supernatant IL-1β (**G**,**H**) and HMGB1 (**I**,**J**) letters refer to CT26 and 4T1 cells, respectively. Untreated cells are representing with CT26 and 4T1.PDT protocols for CT26 cells: PDT1 = 12.2 nM and 25 J/cm<sup>2</sup> ; PDT2 = 31.5 nM and 25 J/cm<sup>2</sup> ; PDT3 = 12.2 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 31.5 nM and 67 J/cm<sup>2</sup> . PDT protocols for 4T1 cells: PDT1 = 9.01 nM and 25 J/cm<sup>2</sup> ; PDT2 = 19.4 nM and 25 J/cm<sup>2</sup> ; PDT3 = 9.01 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 19.4 nM and 67 J/cm<sup>2</sup> . MTX: mitoxantrone; F-T: three cycles of freeze-thawing. Superscripts PER mean the cells submitted to permeation with Triton X-100 0.1%. Equal letters represent results without significant differences between groups. Data are presented as mean ± SEM for triplicates.

**Figure 3.** Assessment of the in vivo immunogenicity of cells treated with different protocols of photodynamic therapy. (**A**) Representation of the vaccination-challenge schedule using CT26 cells and 4T1 cells treated with different PDT-AlPc-NE protocols vs F-T vs MTX. CNTR represent the animal that received just PBS without cells. After the challenge the animals were monitored for: the onset of tumors (**B**,**C**); tumor volume (**D**,**E**); and survival (**F**,**G**) referring CT26 and 4T1, respectively. PDT protocols for CT26 cells—: PDT1 = 12.2 nM and 25 J/cm²; PDT2 = 31.5 nM and 25 J/cm²; PDT3 = 12.2 nM and 67 J/cm²; and PDT4 = 31.5 nM and 67 J/cm². PDT protocols for 4T1 cells: PDT1 = 9.01 nM and 25 J/cm²; PDT2 = 19.4 nM and 25 J/cm²; PDT3 = 9.01 nM and 67 J/cm²; and PDT4 = 19.4 nM and 67 J/cm². 1st vaccine day 0; 2nd vaccine day 10 and the challenge day 17. MTX (mitoxantrone); F-T (three cycles of freeze-thawing); PBS: phosphate buffered saline. Superscript # means *p* < 0.05 in comparison to control group (PBS) at the endpoint. For all data, *n* = 6 mice, mean ± SEM. The CT26 and 4T1 cells have distinct characteristics, such as their origin, immuno-**Figure 3.** Assessment of the in vivo immunogenicity of cells treated with different protocols of photodynamic therapy. (**A**) Representation of the vaccination-challenge schedule using CT26 cells and 4T1 cells treated with different PDT-AlPc-NE protocols vs F-T vs MTX. CNTR represent the animal that received just PBS without cells. After the challenge the animals were monitored for: the onset of tumors (**B**,**C**); tumor volume (**D**,**E**); and survival (**F**,**G**) referring CT26 and 4T1, respectively. PDT protocols for CT26 cells—: PDT1 = 12.2 nM and 25 J/cm<sup>2</sup> ; PDT2 = 31.5 nM and 25 J/cm<sup>2</sup> ; PDT3 = 12.2 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 31.5 nM and 67 J/cm<sup>2</sup> . PDT protocols for 4T1 cells: PDT1 = 9.01 nM and 25 J/cm<sup>2</sup> ; PDT2 = 19.4 nM and 25 J/cm<sup>2</sup> ; PDT3 = 9.01 nM and 67 J/cm<sup>2</sup> ; andPDT4 = 19.4 nM and 67 J/cm<sup>2</sup> . 1st vaccine day 0; 2nd vaccine day 10 and the challenge day 17. MTX (mitoxantrone); F-T (three cycles of freeze-thawing); PBS: phosphate buffered saline. Superscript # means *p* < 0.05 in comparison to control group (PBS) at the endpoint. For all data, *n* = 6 mice, mean ± SEM.

genic profile, and dissemination pattern. The 4T1 murine cells come from a spontaneously originated stage IV breast adenocarcinoma and exhibit a high metastization potential to the lungs, bones, liver, spleen, lymph nodes, and brain[6,32,33]. The CT26 murine cell line is derived from a chemically induced tumor, experimentally developed by the administration of N-nitrous-N-methylurethane [34]. Although CT26 cells can generate lung metastasis, they are less aggressive than the 4T1 cells, which can be a consequence of differences in the efficacy of their respective immunoevasion strategies [6,35,36]. The literature sug-Interestingly, although 4T1 cells exhibited an ICD-related pattern of DAMPs exposure in the in vitro tests described above, they were less immunogenic in vivo than the CT26 cells (Figure 3C). The 4T1 cells treated with PDT or MTX did not elicit a fully protective immunization of the mice; all the animals presented tumors during the experiment. However, there was a significant delay in tumor development in animals vaccinated with MTXor PDT1-treated cells compared to the other groups (Figure 3C,D). Moreover, these miceshowed a prolonged survival time (Figure 3F).

Phenotypically, the 4T1 cells can create an immune-suppressive environment that avoids T cell surveillance, thus protecting tumor cells [6]. The mechanisms are related to the abnormal hematopoiesis process, which is driven to the myeloid lineage that produces

gests that CT26 cells are highly immunogenic [37].

The CT26 and 4T1 cells have distinct characteristics, such as their origin, immunogenic profile, and dissemination pattern. The 4T1 murine cells come from a spontaneously originated stage IV breast adenocarcinoma and exhibit a high metastization potential to the lungs, bones, liver, spleen, lymph nodes, and brain [6,32,33]. The CT26 murine cell line is derived from a chemically induced tumor, experimentally developed by the administration of N-nitrous-N-methylurethane [34]. Although CT26 cells can generate lung metastasis, they are less aggressive than the 4T1 cells, which can be a consequence of differences in the efficacy of their respective immunoevasion strategies [6,35,36]. The literature suggests that CT26 cells are highly immunogenic [37].

Phenotypically, the 4T1 cells can create an immune-suppressive environment that avoids T cell surveillance, thus protecting tumor cells [6]. The mechanisms are related to the abnormal hematopoiesis process, which is driven to the myeloid lineage that produces more myeloid cells in this unusual event. Moreover, due to an excess of growth factors produced by the 4T1 cells, the produced cells are primarily immature cells with immunosuppressive activity. The consequence of this process is an imbalance between the collective memory lymphocytic response, triggered by the vaccination, and the immunosuppressive actions coordinated by the immature immunosuppressive myeloid cells generated after the 4T1 cells stimuli. All these immunological conditions created by the 4T1 cells can explain why the vaccination is less effective in this model compared to the CT26 tumor model.

This study also evaluated the appearance of metastasis foci in the lungs of animals submitted to vaccines with CT26 or 4T1 cells (Figure 4 and Supplementary Materials Figures S6 and S7, respectively). It was found that animals vaccinated with CT26 cells treated with PDT1 had a lung density similar to that of healthy animals, suggesting a low incidence of metastasis (Figure 4). It has already been shown a reduction in CT26 cells metastatic foci in the lungs of mice treated with bacteriochlorin-mediated PDT, an event associated with an immune response induced by PDT against these cells [38]. Regarding the 4T1 cells, a high incidence of metastasis was found in tumor-bearing control mice. However, the radiopacity of the lungs of animals vaccinated with cells treated with PDT1 or MTX was not statistically different from that presented by control, healthy animals (Figure 4B). This result further suggests that, even though these mice (PDT1 and MTX) presented tumors, as discussed earlier, the development of both the grafted tumor and the metastatic foci was somehow reduced. This could be due to an immune response induced in mice by the 4T1 cells succumbing to ICD. Previously, Longo et al. [6] showed that PDT significantly prolonged the survival of mice bearing grafted 4T1 cells tumor, an event associated with a drastic reduction in the number of metastatic foci in the lungs. Moreover, the authors showed that PDT reduced the count of myeloid-derived suppressor cells (MDSC) in the spleen, which can be linked to a reduced ability of 4T1 cells to escape the immunosurveillance and to establish metastasis in PDT-treated mice [6]. That finding could result from the PDT-induced ICD in 4T1 cells, affecting the systemic immune response against tumor cells.

**Figure 4.** In vivo CT quantification of lung density area using HU values. (**A**) Lung density of animals subjected to vaccination with CT26 cells pretreated with PBS (untreated); F-T; MTX and different PDT protocols. (**B**) Lung density of animals subjected to vaccination with 4T1 cells pretreated with PBS (untreated); F-T; MTX and different PDT protocols. Group of healthy animals (naïve) were used as the control for evaluation of lung density (100%-black bars). Lung 2D representative CTtransverse of the animals references the groups: CT26 cells—PDT protocols: PDT1 = 12.2 nM and 25 J/cm²; PDT2 = 31.5 nM and 25 J/cm²; PDT3 = 12.2 nM and 67 J/cm²; and PDT4 = 31.5 nM and 67 J/cm². 4T1 cells—PDT protocols: PDT1 = 9.01 nM and 25 J/cm²; PDT2 = 19.4 nM and 25 J/cm²; PDT3 = 9.01 nM and 67 J/cm²; and PDT4 = 19.4 nM and 67 J/cm². MTX (mitoxantrone); F-T (three cycles of freezethawing); PBS: phosphate buffered saline. Superscript \*, \*\* and \*\*\*\* means *p* < 0.05, *p* < 0.01 and *p* < 0.001, respectively. For all data, *n* = 6 mice, mean ± SEM. **4. Conclusions Figure 4.** In vivo CT quantification of lung density area using HU values. (**A**) Lung density of animals subjected to vaccination with CT26 cells pretreated with PBS (untreated); F-T; MTX and different PDT protocols. (**B**) Lung density of animals subjected to vaccination with 4T1 cells pretreated with PBS (untreated); F-T; MTX and different PDT protocols. Group of healthy animals (naïve) were used as the control for evaluation of lung density (100%-black bars). Lung 2D representative CT-transverse of the animals references the groups: CT26 cells—PDT protocols: PDT1 = 12.2 nM and 25 J/cm<sup>2</sup> ; PDT2 = 31.5 nM and 25 J/cm<sup>2</sup> ; PDT3 = 12.2 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 31.5 nM and 67 J/cm<sup>2</sup> . 4T1 cells—PDT protocols: PDT1 = 9.01 nM and 25 J/cm<sup>2</sup> ; PDT2 = 19.4 nM and 25 J/cm<sup>2</sup> ; PDT3 = 9.01 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 19.4 nM and 67 J/cm<sup>2</sup> . MTX (mitoxantrone); F-T (three cycles of freezethawing); PBS: phosphate buffered saline. Superscript \*, \*\* and \*\*\*\* means *p* < 0.05, *p* < 0.01 and *p* < 0.001, respectively. For all data, *n* = 6 mice, mean ± SEM.

### The present study suggests that the concentration of photosensitizer and the energy **4. Conclusions**

dose are important parameters regarding the ability of PDT to induce ICD in CT26 and 4T1 cells. The application of a milder PDT-AlPc-NE rendered the cells more immunogenic than the more intense PDT protocols. Further studies must address how variations on PDT parameters can affect the in vivo activation of the immune system. The present study suggests that the concentration of photosensitizer and the energy dose are important parameters regarding the ability of PDT to induce ICD in CT26 and 4T1 cells. The application of a milder PDT-AlPc-NE rendered the cells more immunogenic than the more intense PDT protocols. Further studies must address how variations on PDT parameters can affect the in vivo activation of the immune system.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics14010196/s1, Figure S1: Figure S1. Photodynamic effect of AlPc-NE in CT26 cells. The black line represents the CT26 cells exposed to AlPc-NE and maintained in the dark (nonirradiated). The gray line represents the viability of CT26 cells exposed to AlPc-NE for 15 min, washed, left in the dark for different times: (A) 0; (B) 15; (C) 30; (D) 45; (E) 105; and (F) 225 min, respectively. After the incubation, the cells were irradiated for 10 min with a light-emitting diode (LED, λ 660 nm, final energy density of 25 J/cm<sup>2</sup> ). IC50: inhibitory concentrations 50%; IC90: inhibitory concentrations 90%. Data are presented as mean ± SEM for triplicates. Figure S2: Cell viability as a function of the energy density applied (LED, λ 660 nm). The cells were exposed to AlPc IC<sup>50</sup> and irradiated at the specific incubation-to-irradiation times (LED, λ 660 nm) as follows: (A) 0; (B) 15; (C) 30; (D) 45; (E) 105; and (F) 225 min, respectively. Subscript # *p* < 0.01 vs control (100%). Data are presented as mean ± SEM for triplicates. Figure S3. Exposure of calreticulin by CT26 and 4T1 cells exposed to different treatments in vitro. The results for CT26 cells are shown in (A–I) (left panel), and for 4T1 cells in (J–R) (right panel). (A) and (J): Triton X-100 permeabilized cells (TX100); (B) and (K): unpermeabilized control cells; (C) and (L): MTX-treated cells (1.5 µM); (D) and (M): cells subjected to F-T; (E) and (N): PDT1; (F) and (O): PDT2; (G) and (P): PDT3; (H) and (Q): PDT4; (I) and (R): All results for the DAPI marking profile for blue nucleus with green CRT are shown. Data plotted as ± SEM for triplicates. Figure S4. Exposure of HSP70 by CT26 and 4T1 cells exposed to different treatments in vitro. The results for CT26 cells are shown in (A–I) (left panel), and for 4T1 cells in (J–R) (right panel). (A) and (J): Triton X-100 permeabilized cells (TX100); (B) and (K): unpermeabilized control cells; (C) and (L): MTX-treated cells (1.5 µM); (D) and (M): cells subjected to F-T; (E) and (N): PDT1; (F) and (O): PDT2; (G) and (P): PDT3; (H) and (Q): PDT4; (I) and (R): All results for the DAPI marking profile for blue nucleus with green HSP70 are shown. Data plotted as ± SEM for triplicates. Figure S5. Exposure of HSP90 by CT26 and 4T1 cells exposed to different treatments in vitro. The results for CT26 cells are shown in (A–I) (left panel), and for 4T1 cells in (J–R) (right panel). (A) and (J): Triton X-100 permeabilized cells (TX100); (B) and (K): unpermeabilized control cells; (C) and (L): MTX-treated cells (1.5 µM); (D) and (M): cells subjected to F-T; (E) and (N): PDT1; (F) and (O): PDT2; (G) and (P): PDT3; (H) and (Q): PDT4; (I) and (R): All results for the DAPI marking profile for blue nucleus with green HSP90 are shown. Data plotted as ± SEM for triplicates. Figure S6: In vivo computed tomography quantification of the frequency of voxel as a function of HU in the lung. Lung density of animals subjected to vaccination with CT26 cells pretreated: (A) NAÏVE ANIMALS and (A.1) results showed as AUC; (B) PBS (untreated) and (B.1) results showed as AUC AUC; (C) F-T and (C.1) results showed as AUC; (D) MTX and (D.1) results showed as AUC; (E to H) CT26 cells – PDT protocols: PDT1 = 12.2 nM and 25 J/cm<sup>2</sup> ; PDT2 = 31.5 nM and 25 J/cm<sup>2</sup> ; PDT3 = 12.2 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 31.5 nM and 67 J/cm<sup>2</sup> and E.1 to H.1) results showed as AUC, respectively. The assays were performed with a difference of one week (assay 1 to assay 2). For all data, *n* = 6 mice, mean ± SEM. Figure S7. In vivo computed tomography quantification of the frequency of voxel as a function of HU in the lung. Lung density of animals subjected to vaccination with 4T1 cells pretreated: (A) NAÏVE ANIMALS and (A.1) results showed as AUC; (B) PBS (untreated) and (B.1) results showed as AUC AUC; (C) F-T and (C.1) results showed as AUC; (D) MTX and (D.1) results showed as AUC; (E to H) 4T1 cells – PDT protocols: PDT1 = 9.01 nM and 25 J/cm<sup>2</sup> ; PDT2 = 19.4 nM and 25 J/cm<sup>2</sup> ; PDT3 = 9,01 nM and 67 J/cm<sup>2</sup> ; and PDT4 = 19.4 nM and 67 J/cm<sup>2</sup> and E.1 to H.1) results showed as AUC, respectively. The assays were performed with a difference of one week (assay 1 to assay 2). For all data, *n* = 6 mice, mean ± SEM.

**Author Contributions:** Conceptualization, M.C.R. and L.A.M.; Methodology, M.C.R., W.T.d.S.J., T.M., C.L.C.V., J.V.d.O., R.G., T.J.A.P., J.A.V.M., J.P.F.L. and L.A.M.; Formal Analysis, M.C.R., J.P.F.L., R.B.A. and L.A.M.; Investigation, M.C.R., W.T.d.S.J., T.M., C.L.C.V., J.V.d.O., R.G., T.J.A.P., J.A.V.M., J.P.F.L. and L.A.M.; Resources, R.B.A. and L.A.M.; Data Curation, M.C.R. and L.A.M.; Writing—Original Draft Preparation, M.C.R.; Writing—Review and Editing, M.C.R., L.A.M. and J.P.F.L.; Project Administration, L.A.M.; Funding Acquisition, R.B.A. and L.A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Brazilian government agencies FAP-DF/Brazil (0193.001020/ 2015, and 0193.001626/2017), PRONEX FAP-DF, CNPq (0447628/2014-3, 193.001.200/2016) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the Institute of Biology, University of Brasilia (UnB/Doc n. 5529/2015, approved on 3 March 2015).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article or supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest.
