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Effective generation of tumor-infiltrating lymphocyte products from metastatic non-small-cell lung cancer (NSCLC) lesions irrespective of location and previous treatments

Background Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Because current treatment regimens show limited success rates, alternative therapeutic approaches are needed. We recently showed that treatment-naïve, stage I/II primary NSCLC tumors contain a high percentage of tumor-reactive T cells, and that these tumor-reactive T cells can be effectively expanded and used for the generation of autologous tumor-infiltrating T cell (TIL) therapy. Whether these promising findings also hold true for metastatic lesions is unknown yet critical for translation into the clinic. Materials and methods We studied the lymphocyte composition using flow cytometry from 27 metastatic NSCLC lesions obtained from different locations and from patients with different histories of treatment regimens. We determined the expansion capacity of TILs with the clinically approved protocol, and measured their capacity to produce the key pro-inflammatory cytokines interferon-γ, tumor necrosis factor and interleukin 2 and to express CD137 upon co-culture of expanded TILs with the autologous tumor digest. Results The overall number and composition of lymphocyte infiltrates from the various metastatic lesions was by and large comparable to that of early-stage primary NSCLC tumors. We effectively expanded TILs from all metastatic NSCLC lesions to numbers that were compatible with TIL transfusion, irrespective of the location of the metastasis and of the previous treatment. Importantly, 16 of 21 (76%) tested TIL products displayed antitumoral activity, and several contained polyfunctional T cells. Conclusions Metastatic NSCLC lesions constitute a viable source for the generation of tumor-reactive TIL products for therapeutic purposes irrespective of their location and the pre-treatment regimens.


Introduction
Lung cancer is the most common cause of cancer-related mortality world-wide 1 . Non-smallcell lung cancer (NSCLC) accounts for 85% of the patients diagnosed with lung cancer 2 . Several new treatment strategies such as targeted therapy 3,4 , immunotherapy [5][6][7] or a combination of chemo-and immunotherapy have been developed [8][9][10][11] . Nevertheless, the 5-year survival rate for NSCLC patients is still dismal 12 . Therefore, alternative treatment options for late-stage NSCLC patients are required.
Due to their high somatic mutational rate, NSCLC tumors are considered immunogenic 13 .
Indeed, we and others reported a high percentage of T cell infiltrates in NSCLC lesions from treatment-naïve patients [14][15][16] , and evidence that these tumor-infiltrating T cells (TILs) are tumor-specific and cytotoxic is accumulating 17,18 . Adoptive TIL therapy for NSCLC patients could thus represent a viable alternative to current treatment regiments. TIL therapy is based on 4-6 weeks in vitro expansion of autologous TILs derived from tumor lesions that are then re-infused into pre-conditioned patients 19 . In stage III/IV melanoma patients, adoptive TIL therapy achieved an astounding overall response rate of over 50% and a complete response rate of 20% [20][21][22][23] . The similarly high immunogenicity of NSCLC implies that also NSCLC patients could also benefit from TIL therapy.
We previously reported that tumor-reactive TIL products can be efficiently generated from treatment-naïve early-stage primary NSCLC tumor lesions 14 . Importantly, more than 70% of the TIL products contained tumor-reactive T cells that produced at least one of the key proinflammatory cytokines Interferon gamma (IFN-γ), Tumor Necrosis Factor (TNF) and/or Interleukin-2 (IL-2) when exposed to autologous tumor digest 14 . In addition, T cells showed increased expression of the co-stimulatory receptor CD137 (also referred to as 4-1BB) 14 , which is indicative of T cell receptor triggering in this co-culture system 14,24 . Intriguingly, 25% of the expanded TILs were polyfunctional and produced more than one cytokine 14 , a feature that is correlated with the presence of highly functional effector T cells 25,26 . Our study thus demonstrated that tumor-reactive TIL products can be readily generated from treatmentnaive primary NSCLC tumor lesions. Importantly, a recent phase I clinical trial for TIL therapy in late-stage PD-1 refractory NSCLC patients showed striking therapeutic effects 27 . In 11 out of 16 patients (68.8%), tumor regression was observed within one month after treatment 27 . Two patients (12.5%) had a complete response that was ongoing for at least 1,5 years 27 . These exciting results thus highlight the potential of TIL therapy for NSCLC patients. However, to effectively implement TIL therapy for NSCLC patients, it is paramount to define which patient groups are likely to benefit from this treatment. Patients receive are given different treatments (pre-treatment regimen), such as (immuno)chemotherapy, immune checkpoint inhibitors or small molecule inhibitors. Similarly, the location of metastatic tumor lesions 28 may influence the potential of TILs to expand and to respond to autologous tumors. Collecting such data is thus critical for the clinical implementation of TIL therapy for NSCLC patients.
In this study, we provide an in-depth analysis of the lymphocyte composition of metastatic lesions from 27 late-stage NSCLC patients originating from different metastatic locations, and from patients who received various pre-treatment regimens, or were treatment naïve. We show that TILs can be effectively expanded from all metastatic tumor lesions. Importantly, most TIL products contained tumor-reactive T cells, as defined by cytokine production in response to autologous tumor digests. In conclusion, our study demonstrates that tumorreactive TIL products can be generated for therapeutic purposes from late-stage NSCLC lesions, irrespective of the pre-treatment regimen, and the location of the metastatic tumor lesion.

Patient cohort and study design
Between October 2017 and April 2020, 27 late-stage NSCLC patients were included in this study. Characteristics of the patients, origin of metastasis , pre-treatment regimens and time between last treatment and surgery are depicted in Table 1. The cohort consisted of 13 male and 14 female patients between the age of 39 and 87 years (average 60.2 years) with clinical stage III (n=1) and stage IV (n=26) patients according to the TNM7 staging system for NSCLC. 20 patients (74%) had a history of smoking. The study protocol was designed to define the phenotype of TILs isolated from late-stage NSCLC (n=25), to explore the capacity to expand TILs (n=27), and to measure the presence of tumor-reactive T cells within TIL products (n=22).
The study was performed according to the Declaration of Helsinki (seventh revision, 2013), with consent of the Institutional Review Board of the Netherlands Cancer Institute -Antoni van Leeuwenhoek Hospital (NKI-AvL), Amsterdam, the Netherlands. Tumor tissue was obtained directly after surgery, transported at room temperature in RPMI medium 1640 (Gibco) containing 50 µg/ml gentamycin (Sigma-Aldrich), 12,5 µg/ml Fungizone (Amphotericin B, Gibco) and 20% fetal bovine serum (FBS, Bodego), weighed and processed within four hours. Tumor stage and differentiation, weight of the obtained tissues and total tumor weight for each patient are shown in Table 2. Since surgery was not always performed for diagnostic purposes, not all tumor characteristics were determined for each patient. Tumor size ranged from T1a-T4 according to the TNM7 system, and specified as adenocarcinoma (AC, n = 23), squamous carcinoma (SCC, n = 2) and as NSCLC not otherwise specified (NOS, n = 2). On average, 1610 mg tumor tissue was obtained for this study, ranging from 120 -8810 mg.

Tissue digestion
Tissue digestion was performed as previously reported 14 . Briefly, freshly isolated tumorous tissue was finely chopped and incubated for 45 minutes on a rotor at 37°C in RPMI medium 1640 containing 30 IU/ml collagenase IV (Worthington), 12,5 μg/ml DNAse (Roche), and 1% FBS. The digest was pelleted at 360 g for 10 min and resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA). The digest was filtered through a tea mesh and then over a 100 m filter. After red blood cell lysis for 15 minutes at 4°C with 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA (pH 7.4), live and dead cells were manually counted with trypan blue solution (Sigma) on the haemocytometer. 1-2×10 6 live cells were used for flow cytometry analysis, and 1-3×10 6 live cells were used for TIL cultures. The remaining digest was cryopreserved until further use.

T cell expansion
For TIL expansion, 2-3 wells containing 0.5-1 x10 6 live cells from the tissue digest were cultured for 10-13 days in 24 wells plates in 20/80 T-cell mixed media (Miltenyi) containing 5% human serum (HS) (Sanquin), 5% FBS, 50 μg/ml gentamycin, 1.25 μg/ml fungizone, and 6000 IU human recombinant (hr) IL-2 (Proleukin, Novartis) (pre-Rapid Expansion Phase; pre-REP) at 37°C and 5% CO2. Medium was refreshed on day 7, 9 and 11 of the preREP phase. When a monolayer of cells was observed in the entire well, cells were split in 2 wells. When more than 30% of the cells stopped dividing (determined as rounding of cells), cells were harvested, counted and prepared for an additional culture period of 10-13 days (REP). 2×10 5 live cells/well (1-3 wells/patient) were co-cultured with 5-10×10 6 irradiated PBMCs pooled from 15 healthy blood donors in a 24-wells plate, 30 ng/ml anti-CD3 antibody (OKT-3) (Miltenyi Biotec) and 3000 IU/ml hrIL-2. Typically, cells were passaged when a monolayer of cells was observed in the wells during the REP phase (generally every other day) and harvested, washed and counted on day 10-13 after REP, based on visual assessment of the T cell proliferation state as mentioned above. Cells were either used immediately or were cryo-preserved in RPMI medium 1640 containing 10% Dimethyl Sulfoxide (DMSO) (Corning) and 40% FBS until further use.

T cell activation
When enough material was available, fresh or defrosted REP TILs were counted and prestained in FACS buffer with anti-CD4 BUV496 and anti-CD8 BUV805 (BD Biosciences) for 30 minutes at 4°C. Cells were washed twice, i.e. once with FACS buffer, and once with 20/80 Tcell mixed media (Miltenyi). 1×10 5 live expanded TILs were co-cultured with 2×10 5 live tumor digest cells for 6 hours at 37°C. Alternatively, expanded TILs were stimulated with 10 ng/ml PMA and 1 µg/ml Ionomycin (SigmaAldrich), or cultured with T-cell mixed media alone. After 1 hour of co-culture, 1x Brefeldin A (Invitrogen) and 1x Monensin (Invitrogen) were added.

Statistical analysis
Statistical analysis was performed with GraphPad Prism 8.0.2. Compiled data are shown as paired data points for each patient, or as single data points with box and whiskers showing maximum, 75th percentile, median, 25th percentile and minimum, unless otherwise stated in the legend. The overall significance was calculated with unpaired parametric t test with a twotailed p-value, with ordinary 2-way ANOVA test, or with paired parametric t test with a two-tailed p-value, variance was calculated as standard deviation (S.D.). The p-value cut-offs were set on * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.

Late-stage metastatic NSCLC tumor lesions contain high numbers of T cell infiltrates
To determine the rate of viable cells and specifically of T cell infiltrates within metastatic NSCLC tumor tissues, we processed tumor tissues from 27 late-stage NSCLC patients to single cell suspensions with the clinically approved digestion protocol and compared that to our previous study from early-stage primary NSCLC tumors 14 . We collected on average 41.  figure 1(a)), yet displayed a broader range. We therefore assessed if the pre-treatment regimens or the location of the metastatic tumor lesion influenced the T cell content.

Conserved lymphocyte infiltration profile in late-stage metastatic NSCLC lesions
We next questioned how the lymphocyte composition in metastatic NSCLC tumor lesions compared to that of early-stage NSCLC primary tumor lesions (gating strategy: figure 2(a)) 14 .
We therefore measured the percentage of T cell populations, B cells, NK cells and NKT cells.
Overall, the distribution of lymphocyte populations in late-stage metastatic tumor lesions was comparable to that in primary early-stage tumor lesions (figure 2(b)). CD3 + T cells were most prevalent, with 31.8% ± 16.6% CD8 + T cells, 25.1% ± 8.1% conventional CD4 + T cells, and 7.6% ± 5.0% regulatory T cells (Tregs) (figure 2(b)). The percentage of CD8 + T cells and Tregs showed a broader distribution in percentages in metastatic lesions when compared to T cell infiltrates in early-stage primary tumor lesions, yet no significant differences were observed (figure 2(b)).
Only the percentage of conventional CD4 + T cell infiltrates was significantly lower in late-stage NSCLC metastatic lesions when compared to early-stage NSCLC lesions (figure 2(b); p-value: 0.008). Even though trends for various lymphocyte distribution were observed, neither different pre-treatment regiments nor different locations of resected metastases showed overt differences in the distribution of specific lymphocyte subsets (figure 2(c)).
We also determined the ratio between the different T cell subsets (figure 2(d)). Whereas the ratio between CD8 + and regulatory T cells, and between conventional CD4 + and regulatory T cells did not differ between late-stage metastatic and early-stage NSCLC primary tumor lesions, we found a shift in the ratio between CD8 + over conventional CD4 + T cells in late-stage tumor lesions compared to early-stage lesions, with an average ratio of 1.54 (late-stage) compared to 0.8 (early-stage) (figure 2(d); p-value: 0.02). In conclusion, we observed a similar distribution of lymphocyte infiltrates between early-stage and late-stage tumor lesions, except for a reduction of the ratio of conventional CD4 + infiltrates in late-stage NSCLC tumor lesions.

T cells from metastatic lesions display a high memory profile and high expression levels of tissue residency markers
The T cell memory profile is an important indicator for T cell function. In fact, in the early-stage NSCLC cohort, we found high percentages of effector memory T cells 14 . We therefore investigated the memory profile of T cells in metastatic lesions. To this end, we measured the expression of CD27 and CD45RA to distinguish CD27 + CD45RA + naïve (Tnaive), CD27 + CD45RAcentral memory (Tcm), CD27 -CD45RAeffector memory (Tem) and CD27 -CD45RA + terminally differentiated effector memory (Temra) T cells ( figure 3(a,b)). In particular the percentages of CD8 + Tem cells were substantially reduced in late-stage TILs when compared to the early-stage NSCLC TILs (p-value: 0.0003), concomitant with a slight but non-significant increase of percentages of CD8 + Tcm T cells (figure 3(a)). We observed a similar yet non-significant shift towards Tcm away from Tem in the CD4 + T cell compartment (figure 3(b); p-value: 0.09). The percentages of Tnaive or Temra cells in the CD8 + and the conventional CD4 + T cell compartments remained stable ( figure 3(a,b)).

Tissue resident T cells from metastatic lesions express high levels of activation markers
To further define the fitness of the T cell infiltrates from late-stage NSCLC tumor lesions, we measured several exhaustion and activation markers with a multi-color flow cytometry panel.
To further define the activation status of the TILs, we measured the expression of the costimulatory molecule CD137 expression. CD137 expression was shown to enrich for naturally occurring tumor-reactive T cells 39 . In metastatic NSCLC lesions, we found 13.9% ± 9.1% of the tumor infiltrating CD8 + T cells and 6.1% ± 4.0% of the conventional CD4 + T cells expressed CD137 (figure 4(e,f)). Intriguingly, tumor infiltrating CD8 + T cells that express the tissue residency marker CD103 together with CD39 showed with 28.7% ± 14.3% a substantial increase in the percentage of CD137 expression (figure 4(e); p-value < 0.0001). We made similar observations when we used CD69 expression as a marker for tissue residency (figure S3(a)). Albeit not significant, similar trends of increased CD137 expression were detected on CD69 + tissue resident cells conventional CD4 + T cells with or without CD39 expression.
Overall, we measured higher percentages of Tcm cells at the expense of Tem cells in late-stage NSCLC tumor lesions compared to TILs from early-stage NSCLC tumor lesions. Tissue residency markers are increased on CD8 + and CD4 + tumor-infiltrating T cells. Furthermore, the expression of the activation markers CD39 and CD137 are increased, which all combined suggests that tumor-responsive T cells are present in metastatic NSCLC lesions.

Effective expansion of TILs from late-stage NSCLC tumor lesions
We next determined how efficient TILs could be expanded from the metastatic lesions with the clinically approved rapid expansion protocol (REP), the current golden standard for generating TIL products for the clinic 22 . During the first 10-13 days of culturing tumor digests with human recombinant IL-2 (pre-REP), the metastatic TILs expanded on average a 40-fold ( figure 5(a)). The following REP-phase of 10-13 days in the presence of CD3 stimulation and IL-2 resulted in an additional 100-fold expansion of TILs ( figure 5(a)), and a total expansion rate of on average 3500-fold ( figure 5(a)). Late-stage TILs expanded slightly more during the pre-REP phase than early-stage TILs, and less during the REP phase ( figure 5(a)). Yet, the total expansion rate from these two cohorts was comparable ( figure 5(a)) 14 . Furthermore, the expansion rate did not substantially differ for TILs originating from different metastatic locations ( figure 5(b)) or from patients that underwent different pre-treatments ( figure 5(c)).
Also the amount of total viable cells per gram tissue we obtained after expansion is comparable with those we reported for early-stage tumors (figure 5(d)) 14 . Notably, when we used the tumor size (Table 2) figure S4). Conversely, the percentage of CD4 + T cells maintained constant ( figure 5(e)). The ratio of CD8 + T cells over CD4 + T cells increased from a mean ratio of 1.3 ex vivo to 7.6 after REP (figure 5(f); p-value: 0.04). In conclusion, the expanded TIL product display very high T cell content containing both CD4 + TILs and CD8 + TILs yet with a higher proportion of CD8 + TILs.

Tumor-responsive T cells are prevalent in TIL products from late-stage metastatic lesions
Lastly, we studied the functionality of the expanded TILs from metastatic NSCLC lesions. To define the overall capacity of TILs products from late-stage NSCLC tumor lesions, we measured their capacity to produce cytokines upon activation for 6 hours with phorbol myristate acetate (PMA) and ionomycin ( figure S5). Intriguingly, the capacity of individual TIL products to produce TNF, IFN-γ or IL-2 substantially differed, and some TIL products displayed a clear preference to produce one specific cytokine ( figure S5). Nonetheless, each patient produced at least one of the three cytokines to a great extent and most produced two or more ( figure   S5), revealing their capacity to amply produce cytokines.
To define their tumor reactivity, we next examined the responsiveness of the TIL products to the autologous tumor digest, for which enough material was available from 21 patients. As a read-out, we measured the production of TNF, IFN-γ, IL-2 and CD137 (4-1BB) after 6 hours of co-culture with tumor digest, or with medium alone as control (figure 6(a) and figure S6). To distinguish the expanded TILs from T cell infiltrates of the tumor digest, we pre-stained the expanded TILs with CD4 and CD8 antibodies before the co-culture. The generated TIL products contained not only CD8 + T cells, but also CD4 + T cells that produced TNF, IFN-γ, IL-2, or that expressed CD137 in response to tumor digest ( figure 6(b)). The sum of CD8 + T cells that expressed at least one of the cytokines or CD137 (labelled as tumor-responsive) reached 10.0% ± 8.9% in response to tumor digest, compared to 6.6% ± 7.2% positive cells in the medium control (figure 6(c); p-value 0.0007). In CD4 + TILs, the increase was even higher and reached 13.0% ± 8.8% of tumor-responsive cells, compared to 4.5% ± 4.3% positive cells in the medium control (figure 6(c); p-value <0.0001). The level of tumor responsive TILs could not be attributed to a specific location or pre-treatment (figure 6(c)).
We also defined for each TIL product the number of tumor-reactive CD8 + and CD4 + T cells that produced more than one cytokine in response to tumor digest. We divided the cells in single cytokine producers, double producers (i.e. that produce two of the three measured cytokines), and triple producers (i.e. that produce all three measured cytokines) (figure 6(d)). Even though the single cytokine producing T cells contribute most to the tumor-responsive TILs, we observed that almost all TIL products also contained double and triple cytokine-producing TILs, and that the level of polyfunctional TILs were in all cases above background (medium control) (figure 6(d)). Notably, we detected these polyfunctional T cells in 16 out of 21 (76%) TIL products, including those that displayed a lower overall percentage of cytokine-producing T cells (figure 6(d)). Thus, even though a large spread of overall tumor responsiveness is found in TIL products from late-stage metastatic tumor lesions, we observed that tumor responsiveness of TIL products as defined by cytokine production and/or CD137 expression is prevalent.

Discussion
Here, we showed that tumor-reactive TILs can be efficiently isolated and expanded from metastatic NSCLC tumor lesions. This expansion was successful irrespective of the location of the tumor and the pre-treatment regimen the patient received. An important parameter of generating TIL products from metastatic tumor lesions is the presence of T cells. It has been proposed that the origin of tissue from tumors can shape the immune cell composition of metastases 40 . Indeed, the number of CD8 + T cell infiltrates in metastatic renal cell carcinomas and colorectal tumors are comparable to those of the primary tumors 41 . With the exception of brain metastasis from NSCLC tumors 42,43 , which were not included in our cohort, the overall lymphocyte infiltration in metastatic NSCLC lesions also closely resembled those of primary NSCLC tumors 14 . This included high T cell infiltrates with a comparable distribution of CD8 + T

cells, conventional CD4 + T cells and regulatory T cells, and similar percentages of B cells, NK cells, and NKT cells. Based on the high T cell infiltration, we thus conclude that all metastatic
lesion sites tested could serve as tumor source to expand TILs for therapeutic purposes.
In line with their well-established anti-tumoral cytolytic function [44][45][46][47] , the majority of TIL products generated from metastatic NSCLC lesions contained anti-tumoral CD8 + T cell responses, as defined by cytokine production and/or CD137 expression after co-culture with autologous tumor digest. Intriguingly, CD4 + T cells responded even more robustly to autologous tumor digest, a feature that we did not observe in early-stage NSCLC tumors 14 .
Several recent studies identified anti-tumoral cytolytic CD4 + T cell responses in solid tumors, including for NSCLC [48][49][50][51][52] . Therefore, we hypothesize that the observed CD4 + T cell responses could also substantially contribute to T cell responses against NSCLC tumors. Importantly, 16 out of 21 (76%) TIL products contained polyfunctional T cells, which are considered the most potent anti-tumoral T cells 25,26,53 , further implying that the generated TIL products could achieve potential anti-tumoral responses.
Another important point is whether one could identify tumor lesions with a high likelihood of containing tumor-specific T cells already prior to expansion. We and others previously found a correlation between high PD-1 expression on TILs and anti-tumor responses in TIL products 14,54 . However, circulating anti-PD1/PDL-L1 antibodies due to treatment with nivolumab and/or pembrolizumab (anti-PD-1 immunotherapy) impedes such expression analysis for many of the patients in our cohort. However, other markers may help identify tumor-responsive TILs in such a pre-treated patient cohort. We found that the number of tissue resident T cells as defined by expression of tissue resident markers CD103 and CD69 on

CD8 + T cells and by CD69 expression on CD4 + T cells was increased in late-stage tumor lesions.
These tissue-resident TILs showed increased expression of CD39 and were enriched for CD137 expression, which is indicative for TCR engagement and thus potentially for tumor-specific TILs 37,39,55 . CD137 positive T cells were shown to express the highest levels of IFN-γ, TNF, IL-2 39,56,57 , which further points to their capacity to respond to tumor cells. It is therefore tempting to speculate that the combination of these markers, i.e. CD103, CD69, CD39 and CD137 could identify, and potentially even enrich for tumor-specific TILs.
TIL therapy has demonstrated its potential in clinical trials for late-stage melanoma patients 20,22,58 , bladder cancer 59 , breast cancer 60 , ovarian cancer 61 , head and neck cancer 62 , and recently also for NSCLC 27 . In this phase I clinical trial for anti-PD-1 refractory NSCLC patients, 11 out of 16 patients (76%) showed an objective response, and two a complete response, which highlights the potential of TIL therapy for NSCLC patients 27 . Importantly, as for melanoma alike 63 , this study also revealed that TIL therapy can be beneficial for patients that failed immunotherapy. Our results indicate that neither a specific pre-treatment regimen, or the tested locations of metastatic lesions should be a reason to exclude a patient from receiving TIL therapy. However, because the efficacy of TIL therapy is reduced when used as second or third treatment regimen 64 , it may be key for effective treatment to identify which therapy is most suitable for a patient, and to consider TIL therapy as first line treatment.
In conclusion, we showed here that the generation of tumor-reactive TIL products from latestage NSCLC tumor lesions is feasible, irrespective of pre-treatment regimen or tumor origin.
The obtained TIL products reach sufficient cell numbers for clinical application, and most products contain T cells with polyfunctional anti-tumor effector functions. We are therefore confident that TIL therapy will generate promising results for metastatic NSCLC patients in upcoming clinical trials. MCW supervised the study. All authors read and approved the final manuscript.

Acknowledgments
We thank the Flow cytometry facility from Sanquin Research, and the medical assistance staff from the NKI -AvL for technical help.

Conflict of interest
The authors report there are no competing interests to declare.