All in vivo studies were conducted in compliance with Genentech’s Institutional Animal Care and Use Committee in South San Francisco, California, USA. The murine carcinoma cell lines of the colon (CT26)4, breast (JC)5 and lung (TC-1)6 were obtained from American Type Culture Collection (Manassas, Virginia, USA). The murine breast carcinoma EMT6-Luc7 cell line is a variant of the wild-type obtained from ATCC that was engineered in-house to express luciferase. The murine colon carcinoma cell line MC-384 was obtained from Leiden University Medical Center, The Netherlands. All cells were cultured in vitro to confluence and harvested into HBSS:Matrigel (BD Biosciences; Franklin Lakes, New Jersey, USA) (1:1, v:v) for subcutaneous inoculation into syngeneic, immunocompetent female mice (Charles River Laboratories, Hollister, California, USA) aged 8–26 weeks. MC-38 and TC-1 cells were inoculated into C57BL/6 mice whereas CT26, EMT6-Luc and JC cells were inoculated into BALB/c mice. Tumor cell inoculations were made at 0.1 million cells in a volume of 0.1 mL. Naive, age-matched animals were employed as controls for rechallenge inoculations. Tumor growth was monitored with calipers and tumor volumes were calculated from perpendicular length and width measurements using the formula: Tumor Volume (mm³)=0.5 × length × width².

STR was performed under isoflurane anesthesia after tumor volume exceeded 200 mm³ and a minimum of 15 days had elapsed since tumor cell inoculation. Tumors were exposed for excision via opposing elliptical skin incisions made over the tumor and then carefully disconnected from their hosts with a disposable cautery to prevent bleeding. Surgical incisions were closed with wound clips which were removed 7–10 days later. Analgesia was induced immediately prior to STR and maintained thereafter as needed with carprofen (5 mg/kg, s.c.). Animals that experienced primary tumor regrowth due to incomplete STR were not used for subsequent studies.

Draining axillary, brachial and inguinal lymph nodes, spleen and bone marrow were collected from animals determined to be rejecting a 21-day-old secondary tumor which was inoculated >9 months after STR of the primary tumor (CT26, n=10; EMT6-Luc, n=7; MC-38, n=13). Lymph nodes and spleens were gently crushed and disaggregated into a single cell suspension by passing through a 70 µm filter and pooled with bone marrow cells. Pooled material was resuspended for intravenous injection into 10 syngeneic recipients subjected to lethal whole-body irradiation 4–6 hours prior to cell injection. Adoptive transfer was similarly performed from 10 naïve (non-tumor-bearing) donor mice into an equal number of control recipients. Bone marrow ablation in BALB/c and C57BL/6 mice was achieved via one or two exposures to 525 rads staged 4 hours apart using a Cesium-137 irradiator, respectively. Recipients of adoptive transfer were each given 4–10 × 10⁷ cells, corresponding to roughly half the number of cells isolated from each donor mouse. Mice were maintained on medicated drinking water containing glucose (2 g/L), neomycin (1.1 g/L) and polymyxin B (110 mg/L) for 14 days after irradiation. Twenty-one days after bone marrow reconstitution, adoptive transfer recipients were challenged with the standard subcutaneous tumor inoculum.

Draining axillary, brachial and inguinal lymph nodes and spleen were collected from 10 naïve animals as well as from mice bearing subcutaneous MC-38 tumors at 3, 10, 14 and 21 days post a primary tumor cell inoculation (n=10/time point). At each time point tissues were collected, gently crushed and disaggregated into a single cell suspension by passing through a 70 µm filter. Cells were plated at 1×10⁶ per well in complete RPMI-1640 media supplemented with 10% Fetal Bovine Serum, 25 mM HEPES, 4 mM L-Glutamine, 55 µM β-mercaptoethanol (GIBCO) and 10 U/mL of human interleukin-2 (SIGMA). Cells were stimulated by the addition of either dimethyl sulfoxide or a mixture of peptides including the tumor-specific neoantigens M86 and ADPGK as well as p15E (KSPWFTTL, MBL Life Science).8–11 Cells were cultured for 6 hours in the presence of brefeldin A (GolgiPlug, 1 µL/mL, BD Biosciences) to prevent secretion of cytokines. Responding CD8+T cells were identified by flow cytometry using antibodies specific for CD3 (17A2), CD4 (GK1.5) and CD8 (53–6.7). Cells were fixed and permeabilized (CytoFix/Perm, BD Biosciences) prior to staining with anti-interferon-γ (XMG1.2). Samples were analyzed using a BD LSRFortessa X-20 analyzer (BD Biosciences) and FlowJo software (Tree Star).

To test whether growth of a primary tumor is sufficient to lead to the rejection of a tumor rechallenge in the absence of therapy, tumor rejection rates for five commonly used syngeneic tumor models (CT26, EMT6-Luc, JC, MC-38 and TC-1) were assessed 27–81 days following complete surgical resection of an established primary tumor (figure 1B). Rejection of tumor rechallenge after STR was defined as the absence of a palpable mass exhibiting progressive growth for as long as animals were observed after the repeat inoculation, a period lasting more than 7 weeks at a minimum to ensure a delayed onset of tumor growth was not missed as per historical experience with such models. With the exception of animals inoculated with TC-1 tumors, animals that previously harbored CT26, EMT6-Luc, MC-38 or JC rejected a secondary subcutaneous tumor inoculation when the same tumor cell line was reinoculated. The highest incidence of rejection was observed for MC-38 (9/10, 90%) and EMT6-Luc (17/19, 89%), followed by CT26 (11/22, 50%) and JC (6/14, 43%). Rejection rates for primary tumor inoculations was generally less than 10% (CT26, 0%; EMT6-Luc, 4%±4%; JC, 0%; MC-38, 8±5%; TC-1, 2±2%; mean±SEM).

To examine whether tumor-immunity was durable beyond 3 months we assessed the growth on secondary tumor rechallenge more than 9 months following resection of primary tumors (≥310 days CT26; ≥287 days EMT6-Luc; ≥285 days MC-38) (figure 1C). Rejection rates were similar to the rates observed in mice 1–3 months after STR in all models tested with EMT6-Luc showing 100% rejection (7/7), followed by MC-38 (14/21, 67%) and CT26 (11/23, 48%).

We next set out to determine if the anti-tumor immunity seen in these mice could be transferred via transfer of lymphocytes (figure 2). Protection against a primary tumor challenge was afforded to tumor-naïve mice 3 weeks after lethal bone marrow ablation and reconstitution by adoptive transfer of immune cells obtained from the bone marrow, spleen and lymph nodes of mice experiencing tumor rejection 21 days following their repeat inoculation given more than 9 months after excision of their primary tumor. Lymphocytes were transferred from mice following rejection of a secondary tumor to confirm that these mice indeed possessed functional antitumor immunity. Tumor rejection in such adoptive transfer recipients was greater than 70% for CT26 (9/10, 90%), EMT6-Luc (9/10, 90%) and MC-38 (7/10, 70%) far greater than the maximum of 8% spontaneous rejection expected from these cell lines in naïve animals. No protection was afforded to mice that were reconstituted with lymphocytes from non-tumor-bearing controls.

We rationalized if primary tumor inoculation is sufficient to immunize mice to a rechallenge then we should be able to detect the presence of tumor-reactive CD8+T cells in peripheral lymphatic tissues following the first subcutaneous inoculation. To test this, we analyzed CD8+T cells harvested from lymph node and spleen of mice inoculated with MC-38 tumors for their ability to recognize tumor neoantigens (figure 3). CD8+T cells were judged to be tumor reactive if they were capable of producing interferon-gamma on recognition of the well-defined tumor neoantigens present in the MC-38 model: p15E, M86 and ADPGK ex vivo.4–7 Tumor-specific CD8+T cells were detected in pooled spleens and lymph node preps from mice bearing MC-38 tumors 10, 14 and 21 days following inoculation, whereas such T cells were not detected in mice bearing tumors for 3 days nor in naïve control mice.