2.2. RT Immobilizes in the Tumor Microenvironment

To evaluate the spatial distribution of the compound within the tumor, qualitative autoradiography studies were performed. Subcutaneous E0771 tumors were grown in C57/Bl6 mice. Mice were treated with a single intratumoral injection of RT when palpable tumors arose (12 days after implantation), and groups of mice were sacrificed at 24, 72, and 120 h (Figure 4). These images revealed that the compound was able to distribute well throughout the tumor after a single injection, which is evident out to 120 h. To quantitatively evaluate the long-term retention of the RT in the tumor as well as to assess uptake in non-tumor tissues, biodistribution studies were performed with a direct comparison between intratumoral and intravenous administration. RT delivered intratumorally showed high retention in the tumor out to 120 h as well as high tumor to non-tumor ratios, which are ideal for therapy (Figure S2a, Table S1, n = 3). As expected, intravenous administration of the RT resulted in poor tumor retention and high uptake in non-tumor tissues (Figure S2b, Table S2, n = 3).

Open in a separate window

Figure 4

Autoradiography of tumors treated with a single intratumoral injection of RT shows distribution in the TME. C57/Bl6 mice bearing subcutaneous E0771 tumors were treated with a single intratumoral dose of RT (0.15–0.30 MBq). Mice were sacrificed at 24, 72, and 120 h after treatment, and tumors were harvested and flash frozen for autoradiography. Each image represents a slice of an individual tumor. The darker the area, the more radioactive decay that was detected in that area.

2.3. Radiotherapy Results in Improved Prognostic Outcomes

While radionuclide therapy may be administered as a single or fractionated dose in the clinic, studies suggest improved therapeutic efficacy and enhanced antitumor immunity with fractioned regimens, employing intratumoral injections of as little as 2 MBq per dose in murine xenograft models [22,23,24,25]. In an effort to determine the most efficacious dosing regimen for our RT, dose optimization studies were performed. C57/Bl6 mice bearing E0771 subcutaneous tumors on the left flank were treated with RT intratumorally and monitored for overall survival. Preliminary studies utilized a two-dose regimen, with single doses up to 0.74 MBq (Figure S3). This range of radioactivity was insufficient to slow tumor progression, and therefore, the maximum dose was increased to 3.33 MBq and administered as a single dose to monitor host toxicity and tolerability (Figure S4). Mice tolerated treatment well with no acute toxicity seen. Moving to multi-dosing to promote improved efficacy and sustained tumor regression, two doses, with doses ranging from 0 to 4.44 MBq, were given five days apart. This regimen resulted in delayed tumor progression and improved survival outcome (Figure 5).

Open in a separate window

Figure 5

Two administrations of the highest dose RT improved survival outcomes. (A) C57/Bl6 mice bearing subcutaneous E0771 tumors on the left flank were treated with PBS, control (non-radioactive compound), low dose (0.74 MBq), medium dose (2.78 MBq), or high dose (4.44 MBq) on days 1 and 6. *Created using BioRender.com. (B) Tumor volumes were measured every 2–3 days from the start of treatment until mice reached endpoint. Each line represents an individual mouse within the group. (C) Kaplan–Meier survival curves of each group. ** p < 0.01.

2.4. RT + CP Improves Overall Survival in Tumor-Bearing Mice

As CP continues to gain traction as a viable therapeutic option, using PD-L1 expression as a predictive biomarker has become more commonplace. TNBC tumors do indeed express PD-L1 [26,27]; however, the expression is low, and it is not homogenously distributed throughout the tumor, but rather found in focal areas in a small proportion of cancer cells [28]. Further to this, clinical trials have reported both the efficiency and necessity of combined therapeutic modalities, as TNBC patients often have short-lived responses to CP on its own [29]. Based on our preliminary studies, we hypothesized that high-dose RT is capable of sensitizing tumors to CP. Survival studies were performed with the addition of dual CP targeting the non-redundant pathways of cytotoxic T-lymphocyte antigen 4 and programmed death ligand-1 (with anti-CTLA4 and anti-PD-L1 antibodies, respectively). While neither control (non-radioactive TCO-BSA), RT alone, CP alone, or vehicle control + CP showed therapeutic efficacy, the combination of RT + CP resulted in greatly improved overall survival (Figure 6). Mice tolerated treatments well with no toxicity seen.

Open in a separate window

Figure 6

RT + CP significantly improves overall survival. (A) C57/Bl6 mice bearing E0771 tumors were treated with PBS, control, CP (anti-CTLA4 and anti-PD-L1), RT, control + CP or RT + CP. *Created using BioRender.com. (B) Tumor volumes were measured every 2–3 days from the start of treatment until mice reached endpoint. Each line represents an individual mouse within the group. (C) Kaplan–Meier survival curves of each group. ** p < 0.001.

2.5. Radiotherapy + CP Increases TILs in Otherwise Immune-Bare Tumors

To further investigate the impact of each component of our therapy to the TME, histologic assessment was performed. Tumors were harvested on day 7 from mice treated with PBS, control, CP, RT, control + CP, and RT + CP. Analysis of whole tumor sections harvested and stained with hematoxylin and eosin (H&E) shows that mice treated with PBS or the control compound have large tumors with many multi-nucleated cells, suggesting rapid cellular division. Mice treated with CP, RT, or control + CP present with pockets of necrosis and many multi-nucleated cells surrounding these areas, suggesting that although these therapies may induce acute necrosis in areas of the tumor, these mice still have rapid tumor kinetics and disease progression. As expected from survival study outcomes, tumors harvested from mice that were treated with RT + CP present with increased necrosis and shrinking cellular structures, which was likely a direct result of their response to therapy.

Tumors were further stained with CD4, CD8, and F4/80 to assess immune cell infiltrates in the tumor. Mice treated with PBS or the control compound present with moderate levels of CD4⁺ and CD8⁺ cells with densely populated areas of F4/80⁺ macrophages. Mice treated with CP therapy have increased levels of CD4⁺ and CD8⁺ cells and substantial increases of F4/80⁺ macrophages. Interestingly, mice that were treated with RT alone have significantly decreased levels of CD4⁺ and CD8⁺ cells, with a moderate decrease in F4/80⁺ macrophages. Control + CP tumors appeared very similar to those treated with CP alone, suggesting that CP was able to increase the level of CD4⁺, CD8⁺, and F4/80⁺ cells in the tumor. Mice treated with RT + CP have moderate levels of CD8⁺ T cells (similar to PBS treated mice), decreased levels of F4/80⁺ macrophages, and decreased levels of CD4⁺ T cells.

4.2. Chemistry General

Chemicals and reagents for synthesis were purchased from Sigma-Aldrich and Conjuprobe and used without further purification. ¹⁷⁷Lu[Lu] was produced by the McMaster Nuclear Reactor (MNR, Hamilton, Ontario, Canada) using the ¹⁷⁶Lu (p,γ) reaction and was provided as a solution of [¹⁷⁷Lu]LuCl₃ in 0.01 M HCl. Radio-TLC was performed using a Bioscan AR-2000 imaging scanner (West Vancouver, BC, Canada) on iTLC-SG glass microfiber chromatography paper (SGI0001, Agilent Technologies, Santa Clara, CA, USA) plates using 0.1 M EDTA as the eluent. For each TLC performed, plates were spotted with approximately 2 μL (≈3.7 kBq) and run for 5 min. MALDI data were obtained using a Bruker Ultraflextreme spectrometer (Billerica, MA, USA).

4.3. In Vivo Therapy Experiments

Mice were maintained at the McMaster University Central Animal Facility, and all the procedures were performed in full compliance with the Canadian Council on Animal Care and approved by the Animal Research Ethics Board (Animal Utilization Protocol 17-05-22, January 2020) and the Health Physics Department of McMaster University (Permit KM-1, October 2018). Six- to eight-week-old female C57/Bl6 mice (Charles River Laboratories, Wilmington, MA, USA) were used to implant 5 × 10⁶ E0771 cells subcutaneously on the left flank. Mice were weighed and all were found to be approximately 20 g in size. Mice were housed in groups, 5/cage, fed a normal diet, and kept at room temperature. To minimize experimental variability, low-passage E0771 cells were used for subcutaneous injections. Twelve days after injection, the tumors reached treatable average tumor volume (50–100 mm³). Mice were blindly randomized prior to the start of treatment, but not blinded once treatments commenced. In experimental groups receiving control treatment, mice were treated on day 1 and day 5 with DNP-DOTA-BSA (100 µg/50 µL PBS, intratumorally). Experimental groups receiving RT treatment were treated on day 1 and day 5 with ≈4.44 MBq of ¹⁷⁷Lu-DNP-DOTA-BSA (100 µg/50 µL PBS, intratumorally). Experimental groups receiving CP were treated with α-CTLA-4 (BioXCell, BE0131) and α-PD-L1 (BioXCell, BE101) antibodies (200 µg/200 μL PBS each, intraperitoneally) starting on day 3, every 3 days until mice reached endpoint or a total of 10 doses had been given. For all mouse studies, tumors were measured every 2–3 days, and mice having a tumor volume of 1000 mm³ were classified as endpoint.

4.4. Radiochemistry Methods

To a solution of tetrazine, a small molecule (100 µg, 48.0 nmol) in 100 µL of 0.1 M NaOAc (pH 5.5) was added [¹⁷⁷Lu]LuCl₃ (31–74 MBq). The reaction mixture was heated to 60 °C for 5 min, at which point a radio-TLC (cellulose/silica plate) was run in 0.1 M EDTA solution. The radiochemical yield of the reaction was determined to be >99% with >99% radiochemical purity. The radiolabelled tetrazine was added to a solution of TCO-BSA (2 mg/mL) in saline at room temperature for 10 min. The reaction was added to a 50 kDa spin filter and centrifuged at 4000 rpm for 10 min, which had been previously activated with 1.00 mL of saline. The supernatant was washed twice with 1 mL of sterile saline and centrifuged as stated above, which was followed by resuspension in sterile saline for injection. The conjugation efficiency of the reaction was 46 ± 5% (n = 3).

4.5. Autoradiography

C57Bl/6 mice bearing an E0771 flank tumor were administered a single dose of radiolabeled BSA (0.15–0.33 MBq/100 µg, intratumorally) on day 12 of growth when the tumors were palpable (≈100 mm³). The mice were sacrificed after 24, 72, or 120 h (n = 3), at which point the tumors were harvested, placed on a cryomold, and submerged in optimal cutting temperature compound. Then, the cryomold was wrapped in plastic wrap and flash frozen in liquid nitrogen for 15 s. The tumors were sent for analysis, where they were sliced and placed on a phosphor screen for 10 days.

4.6. Biodistribution Studies

Female, 5–6-week-old Balb/c mice ordered from Charles River Laboratory (Kingston, NY) were inoculated with 1 × 10⁶ 4T1 breast cancer cells in the right flank. On day 7 of growth, the mice were administered radiolabeled BSA (0.07–0.33 MBq/100 µg, intratumorally). At 72 and 120 h post-injection (n = 3 per time point), mice were anesthetized with 3% isoflurane and euthanized by cervical dislocation. Blood, adipose, adrenals, bone, brain, gall bladder, heart, kidneys, large intestine and caecum (with contents), liver, lungs, pancreas, skeletal muscle, small intestine (with contents), spleen, stomach (with contents), thyroid/trachea, urine + bladder, tumor, and tail were collected, weighed, and counted in a gamma counter. Decay correction was used to normalize organ activity measurements to time of dose preparation for data calculations with respect to injected dose (i.e., %ID/g).

4.7. Histology

Non-radioactive tumors were resected on day 7, fixed in 10% formalin for 48 h, and then transferred to 70% ethanol for immediate histological processing. Radioactive tumors were resected on day 7, fixed in 10% formalin, decayed for 3 months, and then transferred to 70% ethanol for histological processing. Tumor tissue was embedded in paraffin, and 4-μm sections were prepared. Tissue sections were processed for hematoxylin staining and IHC using Automated Leica Bond Rx stainer with Bond Refine Polymer Detection kit (Leica, DS9800). All antibodies were diluted in IHC/ISH Super Blocker (Leica, PV6199). Primary antibodies and working dilutions using HIER Retrieval Buffer 2 (Leica, AR9640) were as follows: CD3 (1:150; Abcam, ab16669), CD4 (1:800; eBio, 14-9766), and CD8a (1:1000; eBio, 14-0808). For F4/80 (1:500; AbD Serotec, MCA497R), an Enzyme 1 pre-treatment was performed before staining with antibody (AR9551). Antibodies CD4, CD8a, CD19, and F4/80 all required a secondary antibody before polymer detection using Rabbit anti-rat (Vector labs BA4001) at a dilution of 1:100. Immunohistochemistry slides were digitalized using the Olympus VS120-L100-W automated slide scanner (Shinjuku, Tokyo, Japan). They were batch-scanned on the brightfield setting at 20× magnification. The color camera used was the Pike 505C VC50.

4.8. Flow Cytometry Analysis

First, 150 µL blood was collected from the periorbital sinus. Red blood cells from all samples were lysed using ACK buffer. The PBMCs were treated with anti-CD16/CD32 (Fc block) and surface stained with fluorescently conjugated antibodies for FVS (BD Biosciences, #564406), CD4 (BD Biosciences, #561830), CD8 (BD Biosciences, #563046), CD11b (BD Biosciences, #553311), Ly6C (BD Biosciences, #553104), Ly6G (BD Biosciences, #560602), and F4/80 (BD Biosciences, #743282). An LSRFortessa flow cytometer (BD Biosciences, Mississauga, ON, Canada) with FACSDiva software (BD Biosciences, Mississauga, ON, Canada)) was used for data acquisition and FlowJo (Ashland, OR, USA) Mac, version 10.0 software was used for data analysis.

Alyssa Vito was the recipient of the Vanier Canada Graduate Scholarship. The authors would like to acknowledge the core histology facility at McMaster University for their work processing histologic samples and the MPIC Facility for scanning of IHC slides.