PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer

Abstract

Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45^- /CD62P⁺ lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1⁺ lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy.

Keywords: PD-L1; biomarker; extracellular vesicles; immunotherapy; lung cancer.

FIGURE 1

Flow cytometry identifies a panel of tumour antigens expressed on lEVs from NSCLC cell lines. (a+b) Small EVs (sEVs) and large EVs (lEVs) from H2228 and H596 cells were characterized by NTA (a) and transmission electron microscopy (b). Close‐up electron microscopy images of single EVs are provided in Suppl. Figure 1A. (c) Western blot: Expression of common positive and negative EV markers for the two distinct EV populations. The same amount of protein was loaded in every lane (10 μg for upper blot, 15 μg for lower blot). (d) Analysis of lEVs by flow cytometry. Shown is a representative FSC versus SSC plot with the lEV gate used for analysis. PBS only and size bead controls were used to confirm the specificity of the selected gate. (e) Flow cytometry: A two‐fold dilution series of H2228‐lEVs was prepared and the number of events in the lEV gate was recorded during 30 sec of measurement to exclude swarm detection (mean ± SD, n = 3). (f+g) Flow cytometry: Expression of selected tumour antigens (f) as well as PD‐1/PD‐L1 (g) on lEVs from five NSCLC cell lines. Blue: Tumour antigen, grey: isotype control. Shown is one representative histogram out of n = 2 experiments. (h) Expression of PD‐1 was confirmed on lEVs from OCI‐Ly13.2 lymphoma cells. Shown is one representative histogram out of n = 2 experiments.

FIGURE 2

Tumour antigens are highly expressed on lEVs. (a) Expression of the selected nine tumour antigens was compared in cell lysates, lEVs or sEVs from five NSCLC cell lines. Actinin‐4 was included as lEV marker. The same amount of protein was loaded in every lane (20 μg for first blot, 18 μg for second blot, 25 μg for third blot, 17 μg for forth blot). (b) lEVs from the indicated NSCLC cell lines were isolated by differential ultracentrifugation according to the standard protocol and loaded on top of an OptiPrep density gradient (n = 2). The distribution of EV signals and contaminants in the 16 collected fractions was assessed by Western blot. Complete fractions, or 15 μg of protein for cell lysate and plasma sample, respectively, were loaded onto the gel. Actinin‐4 was included as marker for lEVs. Albumin was used to detect serum protein contaminations.

FIGURE 3

NSCLC patients do not show differences in the number or size of lEVs in plasma compared to controls. (a) TEM of lEVs and sEVs isolated from the plasma of a NSCLC patient. The image on the left displays a wide‐field overview of the sample. The panel on the right contains a close‐up image of single vesicles. (b) Representative NTA measurements from plasma‐derived lEVs and sEVs. (c+d) Common lEV markers and contaminants were analyzed on lEVs isolated from NSCLC patients (c) or healthy controls (CTL_h) (d) by western blot. Cell lysates from two NSCLC cell lines as well as a plasma sample are shown as controls. 17 μg of protein were loaded in every lane. (e) NTA: Concentration and size of lEVs in plasma of healthy controls (CTL_h), non‐cancer controls (CTL_nc) and NSCLC patients (median ± 95%CI).

FIGURE 4

Tumour antigen‐loaded lEVs are elevated in the blood of NSCLC patients. (a) Representative scatter plots of plasma‐derived lEVs and platelets. Only events in the lEV gate were used for further analysis. (b) The percentage of lEVs positive for the indicated tumour antigen was measured by flow cytometry in healthy controls (CTL_h), non‐cancer controls (CTL_nc) and NSCLC patients using the gate defined in A. Boxes mark the 25−75 percentiles (line at median) and whiskers the 5–95 percentile. (c) Western blot of EMMPRIN and PD‐L1 expression in lEVs isolated from NSCLC patients and healthy controls. Lysates from H2228 and H596 cells as well as a plasma sample were included as controls. 17 μg of protein were loaded in every lane. (d+e) ROC curves were used to determine the discriminative power of significantly elevated lEV tumour antigens alone (d) or all six combined (e).

FIGURE 5

PD‐L1 is enriched on a population of CD62P⁺/CD45⁻ lEVs in the plasma of NSCLC patients. (a+b) Flow cytometry: Expression of the selected tumour antigens on lEVs from platelets (a) or HDLM2 lymphoma cells (b). CD62P was included as a marker for platelet‐derived lEVs and CD45 for leukocyte‐derived lEVs. Blue: Antigen of interest, grey: isotype control. Shown is one representative histogram out of n = 3 (Platelets) or n = 2 (HDLM2) experiments. (c) The percentage of CD62P⁺ and CD45⁺ lEVs was measured by flow cytometry in healthy controls and NSCLC patients. Boxes mark the 25−75 percentiles (line at median) and whiskers the 5–95 percentile. (d+e) PD‐L1⁺ lEVs were analyzed for their expression of CD62P and CD45. Shown is one representative scatter plot of a healthy control and NSCLC patient (d). The number of PD‐L1⁺/CD62P⁻/CD45⁺ and PD‐L1⁺/CD62P⁺/CD45⁻ lEVs was counted in both groups (e). Boxes mark the 25−75 percentiles (line at median) and whiskers the 5–95 percentile.

FIGURE 6

lEV‐associated PD‐L1 predicts therapy response in patients with absent and low tissue PD‐L1 expression. (a) Kaplan–Meier survival curves of NSCLC patients according to the number of tumour antigen‐positive lEVs in blood. (b) The level of PD‐L1⁺ lEVs in blood of treatment‐naïve patents at initial diagnosis were correlated with response to therapy at the first staging CT after 3 months (R/NR₃) or 6 months (R/NR₆). Based on the staging CT, patients were stratified as responders (R) if they showed complete or partial response or stable disease and as non‐responders (NR) if they showed signs of progressive disease. The upper panel comprises all patients independent of the choice of therapy, the lower panel displays only patients with immunotherapy in their therapeutic regimen. (c+d) Correlation of tPD‐L1 (c) and TPS (d) with the level of PD‐L1⁺ lEVs in plasma. (e) NSCLC patients were stratified according to their tissue PD‐L1 (tPD‐L1) expression. Levels of PD‐L1⁺ lEVs before chemo‐immunotherapy (CIT) alone or in combination with mono‐immunotherapy (ICI) were compared in patients classified as R or NR at the first staging CT after 3 or 6 months of treatment. (f+g) ROC analysis comparing the predictive power of either TPS, tPD‐L1 and PD‐L1⁺ lEVs (f) or of PD‐L1⁺ lEVs in patients grouped by their tPD‐L1 levels (g).