Lung cancer is the leading cause of cancer-related deaths in both men and women with a 5-year survival rate of only 22% [1]. Although these outcomes may be improved by immune checkpoint blockade therapy involving the disruption of the binding of programmed cell death 1 (PD-1, also known as CD279) on tumor-infiltrating lymphocytes (TILs) to programmed death receptor ligand 1 (PD-L1, also known as B7-H1 or CD274) on tumor and tumor-associated cells, most lung cancer patients still fail the therapy, with a response rate of only about 20% [2]. In general, this revolutionary immunotherapy works better against ‘hot’ tumors, which have abundant TILs in the tumor microenvironment, strong immunogenicity and sufficient PD-L1 expression on cancer cells. Unfortunately, most tumors are ‘cold’, with low T-cell infiltration, weak immunogenicity and minimal PD-L1 expression, and show weak response to immune checkpoint inhibitors (ICIs) [3, 4].

Cold lung tumors without targetable oncogenic drivers are treated with chemotherapy as the standard approach [5]. However, the response rate to this conventional cancer therapy is also low and resistance often occurs after an initial response, with an overall survival (OS) of about 12-17 months. Given its roles of inducing TILs and immunogenicity, in particular PD-L1 expression, chemotherapy can be an ideal candidate for combination with PD-1/PD-L1 blockade to improve therapeutic efficacy [6]. Indeed, combination treatment with ICIs and chemotherapeutic drugs shows synergy and better efficacy in both preclinical animal models and clinical trials of lung cancer and other cancers [68]. However, even with the combination of ICIs and chemotherapy, tumors in animals do not remit completely and the objective response rate (ORR) of lung cancer patients only reaches 33%-49.7%, with a median progression-free survival (PFS) of just 5.1-9 months and a median OS of 13-22 months [7, 8]. Thus, further improvement over the chemo-immunotherapy is direly need.

Our recent studies on human and animal lung cancers have shown that most lung tumors not only have low TILs and decreased PD-L1, but also down-regulate major histocompatibility complex class I (MHC-I), thus evading recognition and attack by CD8+ T cells, including those unleashed by ICIs and/or recruited by chemotherapy [6, 9, 10]. Following our initial studies [1117], we have established the PDZ-LIM domain-containing protein PDLIM2, also known as SLIM or mystique [1820], as a bona fide tumor suppressor, and its repression is a causative driver of lung cancer and resistance to ICIs and chemotherapeutic agents [6]. One most important function of PDLIM2 is to promote the ubiquitination and proteasomal degradation of nuclear signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB (NF-κB) RelA (also known as p65), two master transcription factors that function as proto-oncogenes in lung and many other cancers [6, 10-13, 21-27]. Pdlim2 repression in tumor cells thus results in the persistent activation of Stat3 and Rela, leading to MHC-I downregulation and high expression of tumor growth-related genes [6]. In addition, Pdlim2 deficiency also causes strong induction of multi-drug resistance 1 (MDR1) for acquired chemo-resistance [6]. Therefore, PDLIM2 is a potential target to further enhance the efficacy of chemo-and immune-therapies for lung cancers.

In this study, we explored the mechanism of PDLIM2 repression in human lung cancers and tested whether systemic administration of nanoparticle-encapsulated Pdlim2-expression plasmids (nanoPDLIM2) could enhance the efficacy of anti-PD-1 and/or chemotherapeutic drugs in a mouse lung cancer model. These studies indicate that besides epigenetic repression, loss of heterozygosity (LOH) with copy number losses contributes to PDLIM2 downregulation in about 58% of human lung cancers, and that Pdlim2 heterozygous deletion (Pdlim2+/−) mice develop spontaneous tumors in lung and other organs. Notably, systemic administration of nanoPDLIM2 reverses the phenotypes caused by Pdlim2 repression in lung tumors, and its combination with PD-1 and chemotherapeutic drugs shows the strongest therapeutic efficacy compared to single or double therapies, inducing complete remission of lung tumors in 60% of mice. These findings provide a firm basis to target PDLIM2 to enhance the efficacy of ICIs and chemotherapeutic drugs for lung cancer therapy.

Material and Methods

Animals and lung carcinogenesis

We have complied with all relevant ethical regulations for animal testing and research. The animal experiments were performed in accordance with the US National Institutes of Health (NIH) Guidelines on the Use of Laboratory Animals. All animals were maintained under pathogen-free conditions and used according to protocols approved by the Institutional Animal Care and Use Committee (IACAUC) of the University of Pittsburgh. Pdlim2flx/flx/Spc-rtTAtg/−/(tetO)7CMV-Cretg/tg (ΔSPC) mice under a pure FVB/N background and Pdlim2-/- mice under a pure BALB/c background have been described before [6, 19, 21, 22, 28]. For lung carcinogenesis, six-to eight-week-old ΔSPC and wild type FVB/N mice were intraperitoneally (i.p.) injected with urethane (1 mg/g body weight, Sigma-Aldrich, St. Louis, MO, USA) once a week for six consecutive weeks [6], followed by different treatments as shown in the figures. Mice were sacrificed at six weeks post urethane treatment for examination of lung tumors, inflammation, and treatment-induced toxicity. Surface tumors in mouse lungs were counted blinded under a dissecting microscope, and tumor diameters were measured by microcalipers.

Preparation of Pdlim2-expression plasmid or empty vector plasmid nanoparticles

The polyethylenimine (PEI)-based nanoparticles (in vivo-jetPEI) (Polyplus Transfection, New York, NY, USA) and plasmid DNA complexes at a nitrogen-to-phosphate ratio of 8 (N/P=8) were prepared according to the manufacturer’s instructions. Briefly, 25 µg of pCMV-myc-Pdlim2 [21] or empty vector plasmids in 100 μl of a 5% glucose solution were mixed with the in vivo-jetPEI reagent (4 μl) diluted into 100 μl of a 5% glucose solution. After 15 minutes of incubation at room temperature, the mixed solution (200 μl/mouse) was injected intravenously (i.v.) via the tail vein. Presence of nanoPDLIM2 in tissues were measured by PCR assays targeting Amp-R and Lyz2 in the genome of empty vector and Pdlim2 plasmids.

Histology and immunohistochemistry (IHC) analysis

Lung, liver, kidney, and spleen tissues were excised, fixed in formalin, embedded in paraffin, and cut into 4-μm-thick sections. Sections were stained with H&E, or subjected to sequential incubations with the indicated primary antibodies, biotinylated secondary antibodies and streptavidin-HRP [6]. Antibodies were listed in Supplementary Table S1.

In vivo BrdU labeling

Mice were i.p. injected with 50 mg/kg BrdU (Sigma-Aldrich, St. Louis, MO, USA) 24 h prior to sacrifice. Mouse lung tissue sections were stained with anti-BrdU (Sigma-Aldrich, St. Louis, MO, USA). BrdU labeling index was calculated as the percentage of labeled cells per total cells counted (> 500 cells in each counted tumor-containing area).

Flow cytometry (FACS) analysis

The cells were isolated from mouse lungs using dispase digestion followed filtration with 70 μm Nylon cell strainer; then cells were incubated with the antibodies against cell surface antigens after blocked with αCD16/CD32. The cells were then fixed with paraformaldehyde (2%), permeabilized and incubated with antibodies against intracellular antigens if needed. For interferon-γ (IFNγ) staining, cells were treated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml), ionomycin (1 μM), brefeldin A (BFA, 3 μg/ml) and monensin (2 μM) for 4 h before they were stained for FACS analysis. Data were acquired and analyzed by Accuri C6 or LSRFortessa I (BD Biosciences) and FlowJo software [29].

Quantitative polymerase chain reaction (qPCR) analysis

The indicated tissues or cells were subjected to DNA or RNA extraction, RNA reverse transcription and real-time PCR using trizol, reverse transcriptase, and Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA USA) according to manufacturer’s protocol. Primer pairs used for qPCR were listed in Supplementary Table S2.

Microsatellite and gene-specific PCR-based LOH analysis of PDLIM2

Genomic DNAs were isolated from human lung tumors and their matched normal tissues using the PureLink Genomic DNA Purification Kit (Invitrogen, Carlsbad, CA, USA), and subjected to semi- quantitative PCR using the primers specific for the microsatellite markers D8S1786 and D8S1752 that straddle the PDLIM2 genetic locus or the PDLIM2 genetic locus itself. Primer pairs used for the assays were listed in Supplementary Table S2.

Statistical analysis

One-way ANOVA power analysis was used to determine the minimum sample size. Animals were randomly assigned to different treatment groups. Measurements were taken from distinct samples. Student’s t test (two tailed) and one-way ANOVA/Tukey’s or two-way ANOVA/Sidak’s test were used to assess significance of differences between two groups and multiple comparisons, respectively. Pearson’s correlation test was used to assess association between PDLIM2 expression with its promoter methylation or genetic deletion and the overlap between PDLIM2 promoter methylation and genetic deletion. All bars in figures represent means ± standard error of the mean (SEM). The p values are indicated as *p < 0.05, **p < 0.01, ns, not statistically significant, except for those shown in figures. The p values < 0.05 and 0.01 are considered statistically significant and highly statistically significant, respectively. Grubbs’ and ROUT outlier tests were established a priori. There were no exclusions in the analyses.


Both epigenetic repression and genetic deletion of PDLIM2 in lung cancer

Using 40% of the expression level of matched normal lung tissues as the cut-off, PDLIM2 was repressed in over 75% of human lung cancers in a prior study [6]. Analysis of the Cancer Genome Atlas (TCGA) database found that if using 50% as the cut-off, PDLIM2 was repressed in about 93% of human lung cancers (Figure 1A). Further analysis of the TCGA dataset showed that over 70% of human lung cancers with decreased PDLIM2 expression had hypermethylation in PDLIM2 promoter, when using 125% of the methylation level of the PDLIM2 promoter in normal lung tissues as the cut-off (Figure 1A, 1B), validating our previous finding that epigenetic silencing was the main mechanism underlying PDLIM2 repression in lung cancer [6].

PDLIM2 repression in human lung cancers involves both epigenetic alteration and genetic deletion, and PDLIM2 genetic deletion in mice leads to development of spontaneous tumors, majorly lung tumors. (A) TCGA data showing PDLIM2 repression in over 90% of lung cancers if using 50% of the expression level in normal lung tissues as the cut-off (NL, n = 110; Tumor, n = 1019). (B) TCGA data showing PDLIM2 promoter hypermethylation and expression repression (dashed box) in over 70% of lung cancers when using 125% of the methylation level in normal lung tissues as the cut-off (n = 827). (C) TCGA data showing positive associations between PDLIM2 expression and its gene copy numbers as well as PDLIM2 genetic deletion and expression repression (dashed box) in about 58% of lung cancers using the copy number variation of −0.1 as the cut-off (n = 1010). (D) TCGA data showing simultaneous promoter hypermethylation and genomic deletion of PDLIM2 (dashed box) in about 44% of lung cancers (n = 816). (E) Microsatellite-PCR showing pdlim2 loss in human lung cancers (n = 21). (F) qPCR showing PDLIM2 loss in human lung cancer cell lines with known copy number of the PDLIM2 gene (n = 25). (G) Kaplan-Meier tumor-free survival curve showing increased spontaneous tumors in Pdlim2-/- and Pdlim2+/− mice compared to WT mice. Gehan-Breslow-Wilcoxon test was performed. *p < 0.05. (H) Percentage of tumor types spontaneously developed in Pdlim2+/− mice, and 51% of tumors occurred in the lung.

In line with PDLIM2’s location on chromosome 8p21.3, a frequent LOH region in lung and other tumors [3034], analysis of the TCGA database revealed that PDLIM2 expression was positively associated with its gene copy numbers, and that over 58% of human lung cancers with decreased PDLIM2 expression had genetic deletion of the PDLIM2 gene if copy number variation (CNV) of −0.1 was used as the cut-off for the gene deletion (Figure 1C). Further analysis indicated that about 44% of human lung cancers with decreased PDLIM2 expression simultaneously harbored the promoter hypermethylation and LOH with copy number losses of the PDLIM2 gene, and approximately 26% and 14% of them only having the promoter hypermethylation and LOH, respectively (Figure 1D). The gene deletion of PDLIM2 was confirmed by microsatellite and gene-specific PCR-based LOH analysis of human primary lung cancer tissues and cell lines (Figure 1E, 1F; Supplementary Table S3). These data suggested that PDLIM2 repression in lung cancer involves both epigenetic silencing and genetic deletion.

To determine the significance of PDLIM2 LOH in lung tumor, we examined whether Pdlim2 heterozygous deletion leads to decreased Pdlim2 expression and spontaneous tumors in mice. Unlike its absolute absence in Pdlim2 homozygous deletion (Pdlim2-/-) mice, Pdlim2 was detected in the lung of Pdlim2+/− mice, but at a much lower level compared to wild type (WT) mice (about 50% of WT, Supplementary Figure S1A). Importantly, Pdlim2+/− mice, like Pdlim2-/- mice, also developed spontaneous tumors (Figure 1G). Of note, over 50% of tumors developed in Pdlim2-/- and Pdlim2+/− mice were lung tumors (Figure 1H; Supplementary Figure S1B). These data are also highly consistent with the fact that PDLIM2 is ubiquitously expressed under physiological conditions, with the highest level in the lung and lung epithelial cells in particular [6, 18-20]. Thus, PDLIM2 is a haploinsufficient tumor suppressor that is particularly important for lung tumor suppression.

Efficacy of systemic administration of nanoPDLIM2 in refractory lung cancer

Although reversal of PDLIM2 epigenetic repression by epigenetic drugs to restore PDLIM2 expression in cancer cells may be used to treat lung cancers [6], this approach cannot be applied to lung cancers involving PDLIM2 LOH with copy number losses, as occurred in 58% of all lung cancer cases. To overcome this limitation and expand PDLIM2-targeted therapy to all lung cancers with PDLIM2 repression, we tested the therapeutic efficacy of systemic administration of PDLIM2-expression plasmids encapsulated by the clinically feasible in vivo-jetPEI [3539]. To this end, we employed mouse lung cancers induced by urethane, a faithful model of human lung cancer and adenocarcinoma (AC) in particular [6, 23, 24, 40-43]. Urethane is a chemical carcinogen present in fermented food, alcoholic beverage and also cigarette smoke, the predominant risk factor accounting for about 90% of human lung cancers [44]. Like its human counterpart, the murine lung cancer induced by urethane also shares Pdlim2 repression, in addition to their similarities in histology, genetics, molecular biology and immunology [6, 9, 23, 24, 40-43]. WT mice with lung tumors induced by urethane were i.v. injected with nanoparticle-encapsulated Pdlim2 plasmids or empty vector plasmids. Six weeks post the initial treatment of nanoPDLIM2, mice were sacrificed and lung tissues were collected for assays (Figure 2A). Compared to the empty vector plasmid mock-treated group (Vec), nanoPDLIM2-treated mice had a significantly reduced tumor burden (−31%) in their lungs, though the response of tumor numbers was weak (Figure 2B).

Systemic administration of PDLIM2 plasmid nanoparticles shows efficacy in mouse model of refractory lung cancer. (A) Schedule of lung cancer induction and treatment. (B) Intravenous administration of PDLIM2 plasmid nanoparticles reduced lung tumors in mouse urethane lung tumor model (n ≥ 6). Nanoparticles carrying an empty vector plasmid (Vec) that was employed to express PDLIM2 were used as a control. (C) IHC staining showing decreased nuclear expression of Stat 3 and Rela in lung tumors by PDLIM2 nanotherapy (n = 6). (D) IHC staining showing decreased Bcl-xl and increased cleaved caspase −3 in lung tumors by PDLIM2 nanotherapy (n = 6). (E) IHC staining showing decreased Cyclin D1 and proliferation (Brdu) in lung tumors by PDLIM2 nanotherapy (n = 6). Scale bar in (C-E), 20 μm. Student’s t test was performed (two tailed, unpaired) and data represent means ± SEM in (B-E). **p < 0.01; ns, not statistically significant.

In line with the reduction in tumor burden, nanoPDLIM2 administration decreased nuclear Rela and Stat3, a hallmark of NF-κB and STAT3 activation, accordingly reduced the expression of their downstream cell survival gene Bcl2l1(Bcl-xl, −49%) and cell proliferation gene Ccnd1 (Cyclin D1, −61%), increased cleaved caspase 3 (+230%), a marker of apoptosis, and decreased proliferation (−47%) of lung cancer cells (Figure 2C-2E). These data indicated that intravenous administration of nanoPDLIM2 reduced lung tumor burden, suppressed oncogenic Rela and Stat3 activation and increased apoptosis of lung tumors.

High tumor specificity and low toxicity of systemic administration of nanoPDLIM2

To characterize the efficiency of systemically administrated nanoPDLIM2, we examined Pdlim2 expression in lung tumors and several organs, including the liver, kidney and spleen. A high level of Pdlim2 was detected in the lung tumors from mice treated with nanoPDLIM2 one week post the injection, whereas no obvious Pdlim2 was detected in the lung tumors from mice of Vec group (Figure 3A, 3B). It should be pointed out that the tumor delivery efficiency of Pdlim2-expression or empty vector plasmid nanoparticles did not differ (Figure 3B). However, either Pdlim2-expression or empty vector plasmids were hardly detected in other organs/tissues of the same mice, including liver, kidney and spleen (Figure 3B). Of note, in the first two days after injection, both empty vector and Pdlim2 plasmids were also detected at high levels in those tissues but quickly declined afterward (data not shown), consistent with the well-documented tumor-specific enrichment of nanoparticles [37, 45-47]. Consistently, ectopic Pdlim2 was not detected, and the levels of Pdlim2 protein were comparable in these tissues (Figure 3B, 3C).

PDLIM2 nanotherapy shows high tumor specificity and low toxicity. (A) IHC staining showing high Pdlim2 re-expression in lung tumors after PDLIM2 nanotherapy. (B) PCR and IB assays showing lung tumor-specific plasmids delivery and PDLIM2 expression by PDLIM2 nanotherapy (n = 3). (C) IHC staining showing comparable expression of PDLIM2 in the indicated tissues of mice treated with PDLIM2 expression plasmid or empty vector plasmid nanoparticles. (D) No significant changes in animal body weight by PDLIM2nanoparticles (n = 5). (E) H&E staining showing no noticeable changes in major organs by PDLIM2nanoparticles nanotherapy. Scale bar: (A and C) 20 μm; (E) 50 μm.

The i.v. injection of either Pdlim2-expression or control plasmid nanoparticles showed no obvious toxicity to animals, as evidenced by no significant changes in body weight and histology of all organs/tissues examined, including the spleen, kidney and liver (Figure 3D, E). Mouse appearance and behaviors, such as eating, drinking, defecating, urinating, sniffing, grooming and digging, were not different between control and nanoparticle groups (data not shown). Taken together, these data suggest nanoPDLIM2 shows therapeutic efficacy in the mouse lung tumor model while causes low toxicity.

Synergy of nanoPDLIM2 with chemotherapy in lung cancer treatment

Given the role of Pdlim2 in inhibiting the expression of cell survival and proliferation genes in tumor cells, which contribute to chemoresistance, we tested whether nanoPDLIM2 increases the efficacy of chemotherapy in the mouse model of lung cancer. Treatment with carboplatin and paclitaxel, two first-line chemotherapeutic drugs for lung and many other cancers, led to a significant decrease in tumor number (−35%) and tumor burden (−87%) (Figure 4A). Importantly, combination with nanoPDLIM2 further decreased both tumor number (−78%) and tumor burden (−94%), suggesting a promising synergy between nanoPDLIM2 and chemotherapy in lung cancer treatment. Consistently, significantly higher tumor cell apoptosis was detected in mice treated with the combination therapy (+650%), in comparison to those with nanoPDLIM2 (+200%) or chemotherapy (+230%) alone (Figure 4B).

PDLIM2 nanotherapy renders lung cancers more vulnerable to chemotherapy. (A) Synergy of PDLIM2 nanotherapy and chemotherapeutic drugs (carboplatin and paclitaxel) to reduce lung tumors in lung tumor models induced by urethane (n ≥ 5). (B) IHC staining showing effects on lung tumor cell apoptosis by PDLIM2nanoparticle, chemo-drugs, and their combination (n = 6). (C) IHC staining showing increase in nuclear Rela by chemotherapy and its inhibition by PDLIM2nanoparticles (n = 6). (D) IHC staining showing MDR1 induction by chemotherapy and its blockage by PDLIM2 nanotherapy (n = 6). Scale bar in (B-D), 20 μm. Student’s t test was performed (two tailed, unpaired) and data represent means ± SEM. *p < 0.05; **p < 0.01; ns, not statistically significant.

Chemotherapy induced strong Rela activation/nuclear expression and Mdr1 expression (Figure 4C, 4D). NanoPDLIM2 not only repressed the constitutive activation of Rela in cancer cells but also prevented the strong induction of Rela activation and Mdr1 expression by the chemotherapy. Thus, nanoPDLIM2 improves the therapeutic efficacy of chemotherapy through blocking both intrinsic and acquired chemoresistance of lung cancer cells.

NanoPDLIM2 enhanced the efficacy of ICIs in lung cancer treatment

High expression of cell survival genes renders tumor cells resistant to the tumoricidal activity of cytotoxic T lymphocytes (CTLs), including those unleashed by ICIs. Given nanoPDLIM2 administration suppressed survival genes in lung tumors (Figure 2E), we hypothesize it enhances the efficacy of immunotherapy. NanoPDLIM2 increased the number of TILs and the expression of MHC-I, the most important and core components of immunotherapies including PD-1 immune checkpoint blockade therapy (Figure 5A, 5B). In line with our previous studies [6], PD-1 blocking antibody reduced tumor burden (−25%) and tumor number (−13%) (Figure 5C). Combining with PDLIM2 nanotherapy further decreased tumor burden (−64%), and caused a trend toward decrease for tumor number. Consistently, higher increases in both CD4+ and CD8+ TILs, stronger CD8+ CTL activation and lung tumor cell death were detected in combination therapies of nanoPDLIM2 and PD-1 antibody (Figure 5D-5F). These data together suggest that nanoPDLIM2 enhanced the therapeutic efficacy of PD-1 blockade.

PDLIM2 nanotherapy increases the efficacy of PD-1 blockade immunotherapy. (A) IHC staining showing increased TILs in lung tumors by PDLIM2nanoparticles (n = 6). (B) FACS showing increased MHC-I expression in lung tumor cells by PDLIM2 nanotherapy (n = 4). (C) Enhancing effects of PDLIM2nanoparticles on PD-1 immunotherapy in lung tumors induced by urethane (n ≥ 5). (D) IHC staining showing increased TILs by PDLIM2 nanotherapy in the context of immunotherapy (n = 6). (E) FACS showing increased activation of CD8+ T cells by PDLIM2 nanotherapy in the context of immunotherapy (n = 3). (F) IHC staining showing increased lung tumor cell apoptosis by PDLIM2 nanotherapy, immunotherapy, and further increase by their combination (n = 6). Scale bar in (A, D, F), 20 μm. Student’s t test was performed (two tailed, unpaired) and data represent means ± SEM. **p < 0.01; ns, not statistically significant.

Complete remission of all lung cancers in most mice by the combination treatment of nanoPDLIM2 and anti-PD-1 and chemotherapeutic drugs

Like most human lung cancers, lung cancers in our animal model exhibit a decreased expression of PD-L1 on cell surface, in additional to the low expression of MHC-I and low number of TILs [6, 9]. In line with our previous finding that PD-L1 expression is largely independent of Pdlim2 [6], nanoPDLIM2 failed to induce PD-L1 expression in lung tumors; in contrast, chemotherapy induced PD-L1 (Figure 6A). This may explain why the enhancing effect of nanoPDLIM2 on PD-1 blockade therapy is only moderate.

Combination of PDLIM2 nanotherapy, chemotherapy and immunotherapy shows enhanced efficacy in lung cancer treatment. (A) IHC staining showing PD-L1 induction by chemotherapy but not by PDLIM2 nanotherapy. (B) Schedule of lung cancer induction and treatment. (C) Urethane-induced lung tumors were more resistant to chemo and αPD-1 combination therapy in mice with Pdlim2 specifically deleted in lung epithelial cells (ΔSPC) (n ≥ 4). (D) IHC staining showing no MHC-I induction by chemotherapy, PD-1 immunotherapy or their combination (n = 6). (E) Tumor examination showing complete remission of all lung tumors in 60% of mice by combination of the three therapies (n ≥ 5). (F) IHC staining showing increased TILs by PDLIM2 nanotherapy in mice treated with anti-PD-1 and chemotherapeutic drugs (n = 6). (G) FACS analysis showing increased lung CD8+ T-cell activation by PDLIM2 nanotherapy in mice treated with anti-PD-1 and chemotherapeutic drugs (n = 5). (H) No significant effect of PDLIM2 nanotherapy on the body weight of mice treated with anti-PD-1 and chemotherapeutic drugs (n = 5). Scale bar in (A and F), 20 μm. Student’s t test was performed (two tailed, unpaired) and data represent means ± SEM in (c-g). *p < 0.05; **p < 0.01; ns, not statistically significant.

Induction of PD-L1 could protect cancer cells from immune attack and thereby restricts further efficacy improvement from its combination with PDLIM2 nanotherapy, which increased TILs (Figure 4A); on the other hand, upregulated PD-L1 may increase the sensitivity of cancer cells to PD-1 blockade therapy. Indeed, combination of chemotherapeutic drugs and PD-1 blockade showed a promising synergy in reducing both tumor number and tumor burdens in comparison to the individual treatment of chemotherapy or PD-1 blockade therapy [6]. The synergy was largely blocked when Pdlim2 was genetically deleted from lung cancer cells (ΔSPC) (Figure 6B, 6C), suggesting an important role of Pdlim2 in the combination therapy of chemotherapeutic drugs and anti-PD-1. Whereas PDLIM2 nanotherapy increased MHC-I expression on tumor cells (Figure 5B), which is critical for better recognition and killing of tumor cells by CD8+ CTLs, chemotherapy alone or its combination with anti-PD-1 failed to do so (Figure 6D). Accordingly, combination of anti-PD-1 and chemotherapeutic drugs, like the combination therapies of nanoPDLIM2 and anti-PD-1 or chemotherapeutic drugs, also failed to induce a complete remission of lung cancers in any mice.

Given their overlapping roles in increasing TILs and in particular their complement roles in inducing MHC-I and PD-L1 expression on tumor cells, which turn tumors hot and more sensitive to PD-1 blockade, better efficacy is expected for combination of nanoPDLIM2, chemotherapeutic drugs, and anti-PD-1 compared to single therapy or combination of double therapies. Indeed, combination of the three showed the strongest effect on reducing tumor burden and numbers, and caused complete remission of lung cancers in 60% of mice (Figure 6E) and much more shrinkage of lung tumors in remaining mice in comparison to those treated with two combined therapies (data not shown). In line with the increase in therapeutic efficacy, the triple combination therapy significantly increased the numbers and/or activation of CD4+ and CD8+ T cells in the lung, compared to the combination of chemotherapeutic drugs and anti-PD-1 (Figure 6F, 6G).

Of note, consistent with the undetectable toxicity of PDLIM2 nanotherapy, its co-treatment did not further increase the toxicity of anti-PD-1 and chemotherapeutic drugs, as evidenced by no obvious histological difference of major organs, including the liver, lung, kidney, and spleen (Supplementary Figure S2), and no significant differences in animal body weights, in comparison to the mice received the combinational treatment of anti-PD-1 and chemotherapeutic drugs in the presence or absence of empty vector plasmid nanoparticles (Figure 6H).


PD-1/PD-L1 blockade immunotherapy has recently joined chemotherapy as a standard treatment for lung and several other cancers [24]. While some patients have shown dramatic responses, most patients do not benefit from this novel treatment. Currently, various combinations of ICIs with other therapies, in particular with chemotherapeutic drugs, are being extensively tested in both preclinical and clinical trial studies, aiming to expand the benefit of this innovative therapy [68]. Although a promising synergy and better efficacy have been observed in some of these studies [68], further improvement is direly needed. Using a mouse model of lung cancer, we show, for the first time, that PDLIM2 nanotherapy restored Pdlim2 expression in lung tumors and reduced tumor burden; more importantly, co-treatment of nanoPDLIM2 enhanced the efficacy of anti-PD-1 and chemotherapeutic drugs and induced complete remission of all lung cancers in most animals.

Most human lung cancers as well as lung cancers in our animal model have low numbers of TILs and decreased expression of PD-L1 and MHC-I on the cell surface [6, 9], all of which contribute to the resistance to PD-1 blockade therapy. Through inducing immunogenic cell death (ICD) of cancer cells [8], chemotherapy can increase TILs in tumor microenvironment and PD-L1 expression on tumor cells, and thereby synergize with anti-PD-1. However, chemotherapy cannot induce MHC-I expression, this limits further improvement of its synergy with immune therapy for complete cancer remission. Also, tumor cells usually express high levels of survival genes against the tumorcidal effects of chemotherapeutic drugs and of CTLs, including those activated by chemotherapy and unleashed by ICIs.

On the other hand, nanoPDLIM2 induced MHC-I expression and lymphocyte tumor infiltration but did not up-regulate PD-L1. Moreover, nanoPDLIM2 prevented the induction of Mdr1 and the expression of tumor survival genes, this would further sensitize tumor cells to the cytotoxicity of chemotherapeutic drugs and immune cells. Consistent with these, nanoPDLIM2 improved the efficacy of chemotherapy and PD-1 blockade therapy, and the combination of these three therapies resulted in complete cancer remission in 60% of the animals and dramatic tumor reduction in the remaining mice.

Another important clinical characteristic of PDLIM2 nanotherapy is its tumor-specificity and high safety profile. It delivered Pdlim2-expression plasmids specifically to lung tumor tissues and increased Pdlim2 expression. Of note, nanoPDLIM2 showed undetectable toxicity in the animal model, and did not further increase the toxicity of anti-PD-1 and chemotherapeutic drugs. In contrast, although epigenetic drugs (5-aza-dC and MS-275) could restore PDLIM2 expression in cancer cells with PDLIM2 epigenetic repression [6, 10-15] and showed better efficacy as a monotherapy or combined with chemotherapeutic drugs or anti-PD-1 than nanoPDLIM2 (Supplementary Figure S4), the epi-drugs caused body weight loss and much worse toxicity when combined with chemotherapy (Supplementary Figure S3). In addition, the therapeutic efficiency of epigenetic drugs depends on PDLIM2 expression [6]. Although epigenetic therapy can be applicable to 26% lung cancers with PDLIM2 epigenetic repression, it is not suitable in about 58% of lung cancers harboring PDLIM2 LOH with copy number losses. NanoPDLIM2, on the other hand, provides a strategy to restore PDLIM2 expression in all lung cancers with PDLIM2 repression, regardless of whether it is resulted from promoter hypermethylation or gene deletion.


In summary, the presented data identify genetic deletions as a major mechanism for PDLIM2 repression in human lung cancers, and PDLIM2 as a haploinsufficient tumor suppressor particularly important for suppressing lung cancer and therapy resistance. More importantly, these preclinical data demonstrate that combination treatment of nanoPDLIM2, anti-PD-1 and chemotherapeutic drugs induced complete remission of all lung cancers in most animals with high safety profile, suggesting nanoPDLIM2 as a potential strategy to enhance the efficacy of current therapies.


  • AC


  • BFA

    brefeldin A

  • CNV

    copy number variation

  • CTL

    cytotoxic T lymphocyte

  • FACS

    fluorescent activated cell sorting

  • FDA

    Food and Drug Administration

  • IFNγ


  • ICD

    immunogenic cell death

  • ICI

    immune checkpoint inhibitor

  • IHC


  • i.p.


  • i.v.


  • LOH

    loss of heterozygosity

  • Mdr1

    multi-drug resistance 1

  • MHC-I

    major histocompatibility complex class I

  • nanoPDLIM2

    nanoparticle-encapsulated Pdlim2-expression plasmids

  • NF-κB

    nuclear factor-κB

  • NIH

    National Institutes of Health

  • ORR

    objective response rate

  • OS

    overall survival

  • PD-1

    programmed cell death 1

  • PD-L1

    programmed death ligand 1

  • PD-L2

    programmed death ligand 2

  • PDLIM2

    PDZ-LIM domain-containing protein 2

  • PEI


  • PFS

    progression-free survival

  • PMA

    phorbol 12-myristate 13-acetate

  • qPCR

    quantitative polymerase chain reaction

  • SEM

    standard error of the mean

  • STAT3

    signal transducer and activator of transcription 3

  • TCGA

    The Cancer Genome Atlas

  • TIL

    tumor-infiltrating lymphocyte

  • WT

    wild type.


The authors thank Dr. M. J. Grusby (Harvard School of Public Health) and Dr. J. A. Whitsett (University of Cincinnati College of Medicine) for providing PDLIM2-/- and SP-C-rtTAtg/−/(tetO)7CMV-Cretg/tg mice, respectively. The authors also thank Dr. W. Ma and Dr. S. Li (University of Pittsburgh) for their suggestions on the nanoparticle preparation, and Dr. L. H. Rigatti (University of Pittsburgh) for her histological diagnosis of mouse tissues.


This study was financially supported in part by the NIH National Institute of General Medical Sciences (NIGMS) grant R01 GM144890, National Cancer Institute (NCI) grant R01 CA172090, R21 CA259706, American Cancer Society (ACS) Research Scholar grant RSG-19-166-01-TBG, and American Lung Association (ALA) Lung Cancer Discovery Award 821321.

Availability of data and materials

TCGA lung adenocarcinoma, lung squamous cell carcinoma, and lung cancer data we analyzed were obtained from All data generated or analyzed during this study are included in this article and its additional files. The Extra data that support the findings of this study are available from the corresponding authors upon reasonable request.

Author contributions

Z.Q. and G.X. conceived and designed the study, led and contributed to all aspects of the analysis, and wrote the manuscript. S.D.S. contributed to the overall experimental design and data analysis and interpretation, provided advice and constructive feedback, and edited the manuscript. F.S. performed and analyzed all the experimental assays. H.Z. contributed to data analysis and edited the manuscript.

P.Y. originally identified PDLIM2 LOH in human lung cancers by gene-specific PCR-based LOH analysis. Y.X. contributed to mouse clone maintenance and IHC staining.

Antibodies Used

Primers Used

PDLIM2 RNA level and copy number in human lung cancer cell lines

Mice with PDLIM2 deletion develop spontaneous tumors, majorly lung tumors. A, IHC assays showing decreased and complete loss of PDLIM2 protein expression in the lungs of PDLIM2+/− and PDLIM2-/-mice, respectively. Scale bar, 20 µm. B, H&E staining of the lung tissues showing spontaneous lung tumors in PDLIM2+/− and PDLIM2-/-mice. Scale bar, 20 µm.

PDLIM2 nanotherapy causes no obvious pathological changes in major organs. H&E staining showing comparable patterns in lung, liver, kidney and spleen between Vec and PDLIM2 group in the context of combinational chemotherapy and PD-1 blockade immunotherapy (n = 5). Scale bar: 50 µm.

Epigenetic drugs cause body weight loss in mice with lung cancer. Mice were i.p. injected with urethane (1 g/kg) for 6 weeks to induce lung tumors, and then treated for 6 weeks with 5-aza-dC and MS-275 (Epi, i.p., 1 mg/kg each, twice per week), carboplatin and paclitaxel (Chemo, i.p., 30 mg/kg and 15 mg/kg, respectively, once per week), or their combinations. A, Body weight change between prior to the first treatment and the sacrifice endpoint. B, Percentage of mice with peritoneal effusion (n ≥ 4).

Epigenetic drugs show better efficacy than PDLIM2nanoparticle in lung cancer treatment. Mice were i.p. injected with urethane (1 g/kg) for 6 weeks to induce lung tumors, and then treated with 5-aza-dC and MS-275 (Epi, i.p., 1 mg/kg each, twice per week), carboplatin and paclitaxel (Chemo, i.p., 30 mg/kg and 15 mg/kg, respectively, once per week), anti-PD-1 antibody (aPD-1, i.p., 200 µg/mouse, three times per week), PDLIM2-expression plasmids containing nanoparticles (i.v., 25 µg plasmid/mouse, once per week), or their combinations for 6 weeks before they were sacrificed for tumor examination (n ≥ 4).