Inducible caspase 9-mediated suicide gene therapy using AAV6 vectors in a murine model of breast cancer

Breast carcinoma has one of the highest incidence rates (11.7%), with significant clinical heterogeneity. Although conventional chemotherapy and surgical resection are the current standard of care, the resistance and recurrence, after these interventions, necessitate alternate therapeutic approaches. Cancer gene therapy for breast cancer with the suicide gene is an attractive option due to their directed delivery into the tumor. In this study, we have developed a novel treatment strategy against breast cancer with recombinant adeno-associated virus (AAV) serotype 6 vectors carrying a suicide gene, inducible Caspase 9 (iCasp9). Upon treatment with AAV6-iCasp9 vectors and the chemical inducer of dimerizer, AP20187, the viability of murine breast cancer cells (4T1) was significantly reduced to ∼40%–60% (mock control 100%). Following intratumoral delivery of AAV6-iCasp9 vectors in an orthotopic breast cancer mouse model, we observed a significant increase in iCasp9 transgene expression and a significant reduction in tumor growth rate. At the molecular level, immunohistochemical analysis demonstrated subsequent activation of the effector caspase 3 and cellular death. These data highlight the potential of AAV6-iCasp9-based suicide gene therapy for aggressive breast cancer in patients.


INTRODUCTION
The most reported malignancy in females that results in significant mortality is breast carcinoma. 1In 2020, the incidence of breast cancer among all populations is $2.2 million, positioning it first among all cancer types. 2 The global burden of breast cancer is likely to reach $3.19 million by 2040. 2 Clinically, breast cancer is heterogeneous and divided into three major subtypes: estrogen receptor (ER)-positive and progesterone receptor (PR)-positive, human epidermal growth factor receptor-2 + and triple-negative breast cancer (TNBC). 3Drug resistance is a crucial contributing factor mainly associated with the recurrence and relapse of breast cancer. 4Therefore, there is a high demand for supplementing the currently available therapy for breast cancer.In addition to immunotherapy, an effective therapeutic approach is gene therapy, which has shown promise in breast cancer patients at all stages. 4,5ncer gene therapy involves delivering the potential therapeutic transgene to the cancer cells to trigger apoptosis and a bystander ef-fect in the heterogeneous tumor tissue. 6In addition to several gene therapy-based techniques, such as oncogene inhibition, tumor suppressor gene activation, and antiangiogenic therapy, one of the effective methods is suicide gene therapy. 7,8Broadly, suicide gene therapy is categorized into enzyme-activating prodrug therapy and geneinducing cytotoxicity upon drug treatment. 70][11] However, certain limitations, such as the immunogenic nature of the viral and bacterial transgenes, along with the dependency of the activated drug on the cell cycle, can affect the phenotypic rescue. 12An alternative strategy of using an inducible Caspase 9 (iCasp9) transgene, a cytotoxic synthetic analog to mammalian Casp9 gene, linked with an FK506 binding protein (FKBP) of human origin, has been used to silence genes during T cell therapy. 13Chemical inducer of dimerizer, AP20187, a biologically nonreactive small molecule, causes the dimerization of iCasp9 upon binding to the FKBP domain. 14In addition, the cytotoxic effect of the iCasp9/AP20187 combination is not reliant on the cell-cycle phase, which can be useful in a heterogeneous tissue such as breast cancer tissue.A recent study has demonstrated the effectiveness of the iCasp9/AP20187 combination in inducing cytotoxicity in several breast cancer cell lines. 15Adeno-associated virus (AAV) is a highly effective gene delivery vector for treating various monogenic and complex disorders. 16It offers excellent potential in current therapeutic-gene-based delivery in cancer studies because it is nonpathogenic and has low immunogenicity. 17,18][21] Previous reports have highlighted the role of posttranslational modifications on AAV capsid and their impact on vector transduction. 22eddylation is a ubiquitin-like modifier of the target proteins. 23A previous study has shown the involvement of Neddylation in the modulation of the HSV-1 life cycle. 24We have predicted several Neddylation posttranslational modification sites in the capsid protein of AAV2 vectors.Further abolition of these sites resulted in an improved therapeutic outcome during retinal and hepatic gene therapy. 25,26In the present study, we used an AAV6 vector for suicide gene therapy because it has shown significant potential in mitigating the neu-positive murine breast cancer in vivo. 27It must be noted that capsidmodified AAV6 vectors carrying reporter transgenes have been previously reported to have higher transduction in a luminal A type (ER + /PR + ) T47D breast cancer cell line. 28The present study involves the testing and characterization of both the AAV6 wild type (WT) and an AAV6 K31Q Neddylation mutant vector based on the iCasp9 system for suicide gene therapy in a murine model of breast cancer.
78% at different MOI, whereas AAV6K31Q-CAG-iCasp9-treated cells had $40%-58% viability.It is evident that in all AAV6-iCasp9 vector-treated groups, cell viability was significantly (AAV6-CAG-iCasp9: p % 0.05 [all MOIs]; AAV6K31Q-CAG-iCasp9: p % 0.05 [MOI 5 Â 10 3 ], p % 0.01 [MOI 5 Â 10 4 ], p % 0.001 [MOI 1 Â 10 5 ]) decreased compared to mock-treated cells (cell viability 100%).Furthermore, we also observed that the AAV6K31Q Neddylation mutant vector-treated group had very high cell kill ($20%, p % 0.05) when compared to the AAV6 WT vector at different MOI.These results suggested that the AAV6-iCasp9 vectors are able to induce cytotoxicity in 4T1 cells and that the AAV6 K31Q Neddylation mutant vector has higher transduction efficiency in vitro when compared to the AAV6 WT vector, as observed in other AAV Neddylation mutant vectors in our previous studies. 25,26However, it was observed that cell cytotoxicity did not exhibit a proportional increase with increasing vector MOI, which could be a result of a potential saturation dose effect.Once the virus successfully infiltrates the host cell, the subsequent cell death, due to the expression of the suicide gene, becomes independent of the quantity of entering viruses.This implies that only a minimum threshold level of vector presence is required within the cell to trigger the cytotoxic response; any increase in MOI beyond this critical point does not alter the phenotype (cell death).Similar observations were made in a study by Li et al., in which almost no difference was found between MCF-7 cell survival as AAV-2/TRE/HSVtk/Tet-On infection reached a plateau when rAAV titers ranged from 10 4 to 10 6 vector particles/cell. 10

AAV6 vector-mediated suicide gene therapy reduces tumor growth in murine model of breast cancer
We established an orthotopic model of breast cancer in female athymic nu/nu mice.Subsequently, the cytotoxic effect of AAV6-CAG-iCasp9 and AAV6K31Q-CAG-iCasp9 vectors was assessed in these murine models in vivo.After administration of $1 Â 10 6 4T1 cells, the transplanted animals developed visible tumor nodules within the mammary fat pad starting from day 4.After 7 days, when the tumors reached 100-150 mm 3 in volume, $5 Â10 10 vg of AAV6-iCasp9 vectors or PBS in a 100-mL volume was injected intratumorally (Figure 2A).Intraperitoneal injection of AP20187 (1 mg/kg, three doses) was given to each mouse at 48-h intervals starting the next day. 31Mice injected with the AAV6-CAG-iCasp9 and AAV6K31Q-CAG-iCasp9 vectors, without AP20187 intervention, were monitored to assess the exclusive impact of the vectors on tumor growth.
We then periodically assessed the tumor growth rate and volume up to day 10, as recommended earlier. 32Figure 2B depicts the relative tumor growth pattern of the experimental mice.At day 10, the relative tumor volume (RTV) was significantly ($1.35-fold) reduced in AAV6 vector-treated groups when compared to mock-treated animals.The representative data from each experimental group are shown in Figure 2C.The harvested tumors from all five groups are presented in Figure S1.However, no significant difference was observed between AAV6 WT and AAV6 K31Q Neddylation mutant groups (AP20187 administered) in vivo, possibly due to the saturation of transgene expression, as has been observed in other studies. 33,34There was a significant difference in RTV observed (p % 0.001) between AP20187 injected versus noninjected groups.Taken together, the AAV6-iCasp9 vector-mediated (WT and K31Q mutant) suicide gene therapy was well toler-ated and significantly reduced breast cancer tumor growth in vivo in the presence of AP20187.However, a further increased dose of AAV6-iCasp9 vectors via a dose-finding study will be required to completely ablate the breast cancer tumor.

Histological analysis of AAV-treated breast tumor tissue correlates with the formation of apoptotic bodies
Breast tumors were harvested from the orthotropic mice 10 days after vector administration.For morphological analysis, the tumors were fixed, and paraffin sections were taken for H&E staining.Under 100Â magnification, a comparison of cellular morphology was done.Compared to the mock control group, the AAV6-iCasp9-treated tumors (AP20187 administered) had an increased number of apoptotic cells with condensed nuclei (red arrow in Figure 3).Furthermore, the AP20187-treated animals also showed focal areas of necrosis.In contrast, the mocktreated and AAV6-iCasp9 vector-only administered group (without AP20187) had a higher distribution of pleomorphic nuclei (brown arrow), mitotic cells (black arrow), and dense nuclei with higher cell density were seen (Figure 3).Moreover, welldefined features such as cellular debris, shrunken cells, karyorrhectic (marked by yellow circle) and pyknotic nuclei were identified in the AAV6-iCasp9-treated groups (AP20187 administered) but not in the other experimental groups. 35Similar findings have been reported for the iCasp9-based suicide gene therapy in an HCC murine model. 36

AAV6-iCasp9 gene transfer activates the effector caspase in the apoptosis pathway
The major goal of the iCasp9 gene therapy with AAV6 vectors is to trigger the apoptosis pathway.Breast tumors were explanted from all five groups and further homogenized; total RNA was isolated, and cDNA was synthesized.The induction of apoptosis was further confirmed by quantifying the iCasp9 mRNA expression from the cDNA.A qPCR analysis showed that the relative expression of iCasp9 was significantly increased by $6-37 times (p % 0.001) in all AAV6-iCasp9-treated groups (with or without AP20187) when compared to the mock-treated group (Figure S2).Further analysis showed that iCasp9 expression was significantly (p % 0.05) high in the AAV6-iCasp9 capsid mutant-treated group when compared to the AAV6-WT vector-treated group.In addition, tumor sections of all of the AAV6-treated groups were stained with A20 antibody for the detection of intact AAV capsid, and the number of AAV + cells was quantified (Figure 4A). Figure 4B showed a significantly (p % 0.05) high number of AAV-transduced cells in the AAV6K31Q-CAG-iCasp9treated group (39.6 ± 3.3) compared to the AAV6-CAG-iCasp9treated group (28.8 ± 4.6).In addition, we found no detectable AAV + cells in the mock-treated group.The activation of cleaved caspase 3 in the breast tumor sections was then verified by immunohistochemistry (IHC).The cleaved caspase 3 expression was detected in both cytoplasm and nucleus, but the association of cleaved caspase 3 in the nuclei (represented by green arrow) of AAV6-iCasp9 + AP20187-treated groups was higher compared to other groups (Figure 5A).It must be noted that the cleavage and activation of caspase 3 are mediated by induced caspase 9, 37 and further nuclear translocation of cleaved caspase 3 initiates the apoptosis, as observed in our data (Figure 3). 38Further quantification of the activation of cleaved caspase 3 + showed that AAV6-iCasp9 vector-treated groups with AP20187 had significantly (p % 0.001) higher cleaved caspase 3 expression (AAV6-CAG-iCasp9: 21.8 ± 7.5; AAV6K31Q-CAG-iCasp9: 31.6 ± 15.2) when compared to mock-treated and AAV6-iCasp9 vector-only groups (Figure 5B).This signifies that AAV6-iCasp9 vectors failed to activate the caspase 3 in the absence of AP20187, and overexpressed iCasp9 showed functional activity only in the presence of this prodrug.

DNA damage correlates with treatment outcomes in breast tumor
To evaluate the molecular effects of iCasp9, we also performed a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling test (TUNEL).TUNEL assay can detect double-strand breaks in the DNA of apoptotic cells and thus is used as a biomarker for the diagnosis of cell death. 39Tissues from both AAV6-iCasp9-treated groups in the presence of AP20187 were strikingly positive for TUNEL + cells, indicating widespread cell death, which is absent in the AAV6-iCasp9 vector-only group (Figure S3A).To quantify DNA damage, we assessed the fluorescence-integrated density of TUNEL + cells.Data depicted in Figure S3B reveals TUNEL + cells with higher fluorescence-integrated density in tumors that received suicide gene therapy (AAV6-CAG-iCasp9: 1.7 Â 10 5 and AAV6K31Q-CAG-iCasp9: 2 Â 10 5 per unit area, p % 0.05 and p % 0.001, respectively) when compared to the mock-treated group.These data highlight that the AAV6K31Q mutant is effective in triggering apoptosis in the breast cancer cells in the presence of AP20187.1][42] Although we noticed high AAV transduction in the mutant group, the same does not get reflected in terms of RTV, and we speculate that this discrepancy may stem from the limited availability of the dimerizer drug AP20187, which is integral to the ultimate cell kill effect.Future investigations focusing on dose optimization of AP20187 could help elucidate its impact on RTV.
To assess the off-target effect of iCasp9 activation in normal organs (e.g., liver) of AAV6-iCasp9-treated animals, IHC of caspase 9 protein was performed.Figure S4 demonstrated the absence of caspase 9 expression in the liver of AAV vector-administered groups.This implies that this direct intratumoral suicide gene therapy had a negligible effect on normal tissues.

DISCUSSION
TNBC has an increased resistance against available chemotherapies such as taxane and anthracycline-based drugs, emphasizing the critical need for research into novel strategies to slow disease progression. 43,44Gene therapy, by virtue of its direct mechanism of triggering apoptosis in the target cell, is an effective approach for cancer treatment. 45In particular, gene therapy using an AAV vector in breast cancer has shown encouraging results.A study was conducted in the NeuT breast cancer model with a septuplet-tyrosine mutant form of AAV2 vector-mediated delivery of small interfering RNAs (siRNAs) against proteins involved in unfolded protein response. 46he outcome data showed that treated xenograft tumors had decreased growth and angiogenesis.In another study, suicide gene therapy by intratumoral administration of doxycycline-inducible AAV2-HSV-TK resulted in substantial suppression of tumor growth in a mouse model of breast cancer (MCF-7). 10In a study of immunomodulation of neu-expressing Turin-Bologna breast cancer tumors, AAV5-neu or AAV6-neu expression increased the survival (80% versus 100%, up to day 300) of the animal model by inducing long-term immunity against the tumor. 27Pinto et al. showed that the transduction of MDA-MB-468 cells with an AAV2-short hairpin RNA vector directed against proteasome subunit alpha-2 caused a dramatic reduction in cell survivability (from 100% to 30%) and an increase in the induction of apoptosis, by a factor of two. 47AAV2-mediated overexpression of high-affinity vascular endothelial growth factor blocker and paclitaxel improved therapeutic response in a murine model of TNBC. 48Here, for the first time, we have demonstrated the therapeutic potency of the iCasp9 transgene in the murine TNBC model. 49e inducible caspase 9-based system offers several benefits compared to enzyme-activated prodrug therapies, as highlighted earlier. 201][52] Second, the cell-cycle dependency of enzyme-activated prodrug therapies limits their cytotoxic effects to the actively dividing cells only, which may decrease their therapeutic potential in a heterogeneous tumor tissue consisting of senescence cancer stem cells. 53,54Upon the administration of AAV6-iCasp9 vectors, caspase 3 and other components of the intrinsic apoptotic pathway are activated after the dimerization of iCasp9 protein by AP20187. 55,56This activation of the proapoptotic pathway occurs directly and is not dependent on cell-cycle phase. 57ur in vivo study has demonstrated that after administering the AAV6-iCasp9 vector/AP20187 combination, the breast tumors had a lesser tumor volume and growth rate within 6-10 days of vector administration.In comparison, previous studies in breast cancer models showed retarded tumor growth after 30 days of administration of HSV-TK/ganciclovir-based gene therapy. 10The induction of apoptosis was also further validated by ex vivo analysis of breast tumor tissue.Although we have observed the therapeutic potential of our iCasp9 gene at the cellular level, despite having breast tumor growth retardation, tumors were not completely eradicated.This suggests that monotherapy may not be sufficient, and further enhancement by using an adjunct or combinatorial treatment with chemotherapeutic or immunotherapeutic drugs is required.
Our study has the following limitations.We have used a strong ubiquitous CAG promoter for the expression of iCasp9 transgene.The therapy will be more beneficial if further tumor-targeting strategies at the transcription or vector transduction levels can be applied.Instead of using ubiquitous promoters, using a cancer-specific or tumor-specific promoter is desirable. 58,59Yun et al. have shown that promoters of the protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) genes can be used in targeting breast cancer cells because these promoters exhibit strong gene expression similar to that driven by the CMV promoter. 60At the transduction level, the AAV vector can be modified by peptide insertion and then targeted towards specific cancer cells.A previous study from our lab showed a modified AAV6 vector carrying CD33 targeting peptide against human myeloid leukemia cells (U937). 21Another strategy to augment the effectivity of iCasp9-based gene therapy could be the modulation of hallmark parameters of the tumor microenvironment, such as hypoxia, known to confer resistance to certain forms of therapy. 61Therefore, enhancing the iCasp9 system by altering the regulatory components of the transgenic construct, increasing the effective vector dose from the currently used $5 Â 10 10 vg by a log fold, or combining it with other adjunct therapies may pave the way for its clinical translation in patients with breast cancer.
(Cambridge, MA) and was reconstituted in 100% ethanol at a 1-mM concentration and kept at À20 C.

Prediction of Neddylation sites on AAV6 capsid
Amino acids for Neddylation were predicted in the VP1 protein of AAV6 using the sequence of the VP1 protein (Protein ID: AAB95450.1).NeddyPreddy, a web-based bioinformatics tool, was used to predict potential sites for Neddylation. 62The output was used to determine the medium and high threshold levels for this tool.The AAV6 K31Q target was selected based on the high threshold score of 0.9 out of 1.The Neddylation mutation (K31Q) in the AAV6 WT rep/cap plasmid (p.AAVR2/C6) was further generated by sitedirected mutagenesis using specific primers.

Titration for quantification of AAV vector genome
The physical particle titer of AAV vectors was measured.The sample was treated with DNase to remove free DNA.A qPCR with SYBR Green (Promega, Madison, WI) and polyadenylation (PolyA) signal-specific primer was carried out in CFX96 (Bio-Rad, Hercules, CA).AAV2-RSS (American Type Culture Collection, Manassas, VA) was used as a standard for titration.Titers were measured in vg/mL and calculated from two independent analyses.

In vitro cytotoxicity assay
For the comparison of cytotoxicity by AAV6-CAG-iCasp9 and AAV6K31Q-CAG-iCasp9 vectors, $1.5 Â 10 4 4T1 cells were seeded in triplicate and transduced at MOIs of 5 Â 10 3 , 5 Â 10 4 , and 1 Â 10 5 .Vector-transduced cells were treated with 10 nM AP20187 the next day.An absorbance-based EZcountTM MTT Cell Assay Kit (HiMedia, Mumbai, India) was used to evaluate cell cytotoxicity 24 h after drug treatment.The percentage of cell survivability was calculated as described by the manufacturer protocol. 10ducible Casp9 gene therapy in breast cancer murine model Female athymic nu/nu mice (National Institute of Nutrition, Hyderabad, India) were used for this study.Animal experiments were conducted following the approval of the IIT-Kanpur institutional animal ethics committee.For orthotopic injection in 6-to 8-week-old female athymic nu/nu mice, $1 Â 10 6 4T1 cells were resuspended in 100 mL filtered PBS and administered in the sixth mammary fat pad region.Upon reaching a tumor volume of $100-150 mm 3 , mice received $5 Â 10 10 vg of AAV6-CAG-iCasp9 or AAV6K31Q-CAG-iCasp9 vectors intratumorally. 20Approximately 0.1 mL of PBS was injected to mimic the injection process in the mock group.After administering the vector, the animals received 3 doses of intraperitoneal AP20187 (1 mg/kg) injections at 48-h intervals starting the next day of vector administration.The control groups included two treatment groups that only received the AAV6-CAG-iCasp9 or AAV6K31Q-CAG-iCasp9 vectors and a third group receiving the sham intraperitoneal injection to observe the AAV6-iCasp9 vector-only effect in the tumor development.Tumor diameter was measured every alternate day using a Vernier caliper at two perpendicular diameters in the experimental mice.The animals were examined visually for any other gross changes daily.Tumor volume was calculated using the equation 0.5 Â L Â W 2 , where L and W are the longest and the smallest diameter (mm), respectively. 64RTV was calculated by dividing tumor volume at a particular day (T X ) by tumor volume at day 0 (T 0 ), where RTV T = RTV of the treated group and RTV M = RTV of the mock group. 65

Gene expression quantification
Total RNA was isolated from the breast tumor tissue of each group.Approximately 50 mg of tissue was first homogenized using IG-L13K Mini Handheld Homogenizer (iGene Labserve, New Delhi, India), and further RNA isolation was done with Trizol (Invitrogen, Waltham, MA) reagent.Isolated RNA was run in a 1.5% agarose gel (with 1% sodium hypochlorite), and the integrity of 28S, 18S, and 5S rRNA was checked.For analysis of iCasp9 mRNA expression, a qPCR assay with SYBR Green (Promega) was performed (3-6 technical replicates per group).The primer sequence was 5 0 -GAAGGG GTTGCCCAGATGAG-3 0 (forward) and 5 0 -GCACCGACATCACC AAATCC-3 0 (reverse).The relative normalized expression of iCasp9 was plotted using 18S rRNA expression as a reference for each sample.

Histopathological studies
Tumors from mock and AAV-treated groups were harvested after 10 days of AAV vector administration, washed in PBS, and fixed in 10% neutral buffered formalin at room temperature for 24 h.Tumor tissues were further processed by paraffin embedding, tissue sectioning, and H&E staining.Thereafter, stained sections were analyzed by microscopic examination. 66Images were acquired under an inverted microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany) at 100Â magnification.

In situ cell death detection
Paraffin blocks containing tumor tissue were cut using a microtome (Leica Biosystems) to obtain tumor tissue sections.Following this, the 8-mm sections (n = 6-8) were processed through a TUNEL assay following the manufacturer's protocol (Roche, Basel, Switzerland) to detect cells undergoing DNA damage and apoptosis. 39Additional washing steps were followed by nuclear staining with DAPI (1:1,000; Thermo Fisher, Waltham, MA) and mounting (FluorSave Reagent; Millipore, Merck, Burlington, MA).Imaging was performed using a confocal microscope (LSM780NLO, Carl Zeiss GmbH, Wein, Austria).Micrographs were acquired at a 40Â magnification.The fluorescence-integrated density was measured using ImageJ (NIH, Bethesda, MD) software. 67

Statistical analysis
Statistical significance was evaluated by an unpaired t test with Welch's correction or a two-way ANOVA with Bonferroni correction, as appropriate (GraphPad Prism 8.0 Software, La Jolla, CA).

Figure 2 .
Figure 2. Antitumorigenic effect of AAV6-iCasp9 vectors (WT and K31Q mutant) with AP20187 dimerizer drug in an orthotopic model of breast cancer (A) An orthotopic allograft breast cancer model was developed by administering the 4T1 cell line into the mammary fat pad of female athymic nu/nu mice.When the tumor size reached a volume of $100-150 mm 3 , AAV vectors were administered intratumorally.On days 2, 4, and 6 post-vector administration, AP20187 was administered intraperitoneally.Tumors were harvested at day 10 post-vector administration for further molecular and biochemical assays.**p % 0.01; ***p % 0.001 versus mock group; # p % 0.05, ### p % 0.001 versus AAV6-CAG-iCasp9; &&& p % 0.001 versus AAV6K31Q-CAG-iCasp9. (B) Animals from the AAV6-CAG-iCasp9 and AAV6K31Q-CAG-iCasp9 groups with AP20187 treatment had a more attenuated tumor growth rate than mock-injected and AAV6-iCasp9 vector-only animals (p % 0.001).Data are represented as mean ± SD. (C) Representative animals at day 10 from each experimental group are depicted.

Figure 3 .
Figure 3. Histological analysis of tumors in breast cancer allograft tissueHistological comparison among all of the groups depicted that AAV6-iCasp9 +AP20187-treated groups had focal areas of necrosis, pyknosis, karyorrhexis (marked by yellow circle), and apoptotic bodies (shown by red arrow) than the mock-treated and AAV6-iCasp9 vector-only groups (magnification 100Â; scale bar, 25 mm), whereas more numbers of pleomorphic nuclei (marked by brown arrow) and mitotic cells (marked by black arrow) were observed in mock-treated and AAV6-iCasp9 vector-only groups.The data was generated from 2 representative tumors from each group and a total of 6 sections from each group.

Figure 4 .
Figure 4. Detection of AAV particles in breast cancer tissue Tumors from each treatment group were analyzed by IHC with A20 antibody specific to detect AAV capsid.(A) Micrographs from the AAV-treated groups with or without AP20187 had a clear distribution of AAV + cells within the tumors when compared to mock-treated animals (III, magnification 63Â; scale bar, 50 mm).The magnified regions of (III) are represented in (IV) (scale bar, 25 mm).AAV + cells are marked with green arrows.(B) Quantification using ImageJ showed that the number of AAV + cells was higher in the AAV6K31Q-CAG-iCasp9-treated group compared to the AAV6-CAG-iCasp9-treated group (***p % 0.001 versus mock group; # p % 0.05 versus AAV6-CAG-iCasp9 + AP20187).Data are represented as mean ± SD.The data are generated from 5 sections from each treatment group.