Tumor Hypoxia Imaging Research Articles

A hypoxia-targeting and hypoxia-responsive nano-probe for tumor detection and early diagnosis.

Accurate imaging of tumor hypoxia in vivo is critical for early cancer diagnosis and clinical outcomes, highlighting the great need for its detection specificity and sensitivity. In this report, we propose a probe (HTRNP) that simultaneously has hypoxia-targeting and hypoxia-responsive capabilities to enhance the tumor hypoxia imaging efficiency. HTRNP was successfully prepared through the encapsulation of Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP), which exhibits hypoxia-dependent phosphorescence, within the amphiphilic block copolymer OPDMA-PF, which has hypoxia-targeting tertiary amine N-oxide moieties and hydrophobic perfluorobenzene ring structures, which highly improved the loading content and water solubility of PtPFPP. By combining targeting and response abilities toward hypoxic conditions, the HTRNP micelles efficiently accumulate in the tumor tissues and emit intense phosphorescence, thus enabling ultrasensitive detection of various tumor models, even of hundreds of cancer cells, indicating its promising potential for early cancer detection and phenotypic characterization.

Enzyme-induced liquid-to-solid phase transition of a mitochondria-targeted AIEgen in cancer theranostics.

Enzyme-instructed self-assembly (EISA) is a promising approach to anti-cancer therapeutics due to its precise targeting and unique cell death mechanism. In this study, we introduce a small molecule, DN6, which undergoes nitroreductase (NTR)-responsive liquid-liquid phase separation (LLPS) followed by a liquid-to-solid phase transition (LST) through a gel-like intermediate state, resulting in the formation of nanoaggregates with spatiotemporal control. The reduced form of DN6 (DN6R), owing to its aggregation-induced emission (AIE) and mitochondria-targeting capabilities, has been employed for organelle-specific imaging of tumor hypoxia. The red-emissive DN6R nanoaggregates in situ generated by NTR induce mitochondrial damage and oxidative stress, culminating in apoptosis in cancer cells and spheroids. The organelle-specific targeting, visualization, and therapeutic outcomes achieved by leveraging LST of NTR-responsive AIEgenic DN6 render it as a promising agent for cancer theranostics.

Structural insights into the enzymatic breakdown of azomycin-derived antibiotics by 2-nitroimdazole hydrolase (NnhA)

The antibiotic 2-nitroimidazole (2NI) or azomycin, used for treating drug-resistant tuberculosis and imaging tumor hypoxia, requires activation by bacterial nitroreductases for its antibiotic and cytotoxic effect. Mycobacterium sp. JS330 produces 2-nitroimidazole nitrohydrolase (NnhA) that circumvents 2NI activation, conferring 2NI resistance by hydrolysing it to nitrite and imidazol-2-one (IM2O) instead. This study elucidates NnhA’s structure, catalytic mechanism, and evolutionary background within the guanidino-group modifying enzyme (GME) superfamily, aided by a more soluble protein variant engineered through directed evolution. Despite low sequence similarity and limited occurrence in a few soil-dwelling mycobacteria and Actinomycetota, NnhA maintains the α/β propeller fold characteristic of GME superfamily enzymes and forms an unusual hexameric ring structure formed by a trimer of domain-swapped dimers. The similarity of its active site to arginine deiminases (ADIs) and human dimethylarginine dimethylaminohydrolases (DDAHs), along with molecular dynamics simulations, suggests NnhA’s catalytic mechanism resembles the hydrolysis reactions of these related enzymes.

A thiourea-bridged 99mTc(CO)3-dipicolylamine-2-nitroimidazole complex for targeting tumor hypoxia: Utilizing metabolizable thiourea-bridge to improve pharmacokinetics.

The 2-nitroimidazole based 99mTc-radiopharmaceuticals are widely explored for imaging tumor hypoxia. Radiopharmaceuticals for targeting hypoxia are often lipophilic and therefore, show significant uptake in liver and other vital organs. In this context, lipophilic radiopharmaceuticals with design features enabling faster clearance from liver may be more desirable. A dipicolylamine-NCS bifunctional chelator that could generate a thiourea-bridge up on conjugation to primary amine bearing molecule was used to synthesize a 2-nitroimidazole-dipicolyl amine ligand for radiolabeling with 99mTc(CO)3 core. Corresponding Re(CO)3-analogue was prepared to establish the structure of 2-nitroimidazole-99mTc(CO)3 complex prepared in trace level. The 2-nitroimidazole-99mTc(CO)3 complex showed a hypoxic to normoxic ratio of ~2.5 in CHO cells at 3 h. In vivo, the complex showed accumulation and retention in tumor with high tumor to blood and tumor to muscle ratio. The study demonstrated the utility of metabolizable thiourea-bridge in 2-nitroimidazole-99mTc(CO)3 complex in inducing faster clearance of the radiotracer from liver. The dipicolylamine-NCS bifunctional chelator reported herein can also be used for radiolabeling other class of target specific molecules with 99mTc(CO)3 core.

Recent Advances in the Application of Nitro(het)aromatic Compounds for Treating and/or Fluorescent Imaging of Tumor Hypoxia.

This review delves into recent advancements in the field of nitro(het)aromatic bioreductive agents tailored for hypoxic environments. These compounds are designed to exploit the low-oxygen conditions typically found in solid tumors, making them promising candidates for targeted cancer therapies. Initially, this review focused on their role as gene-directed enzyme prodrugs, which are inert until activated by specific enzymes within tumor cells. Upon activation, these prodrugs undergo chemical transformations that convert them into potent cytotoxic agents, selectively targeting cancerous tissue while sparing healthy cells. Additionally, this review discusses recent developments in prodrug conjugates containing nitro(het)aromatic moieties, designed to activate under low-oxygen conditions within tumors. This approach enhances their efficacy and specificity in cancer treatment. Furthermore, this review covers innovative research on using nitro(het)aromatic compounds as fluorescent probes for imaging hypoxic tumors. These probes enable non-invasive visualization of low-oxygen regions within tumors, providing valuable insights for the diagnosis, treatment planning, and monitoring of therapeutic responses. We hope this review will inspire researchers to design and synthesize improved compounds for selective cancer treatment and early diagnostics.

Assessment of tumor hypoxia in spontaneous canine tumors after treatment with OMX, a novel H-NOX oxygen carrier, with [18F]FMISO PET/CT

BackgroundHypoxia is a detrimental factor in solid tumors, leading to aggressiveness and therapy resistance. OMX, a tunable oxygen carrier from the heme nitric oxide/oxygen-binding (H-NOX) protein family, has the potential to reduce tumor hypoxia. [18F]Fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) is the most widely used and investigated method for non-invasive imaging of tumor hypoxia. In this study, we used [18F]FMISO PET/CT (computed tomography) to assess the effect of OMX on tumor hypoxia in spontaneous canine tumors.ResultsThirteen canine patients with various tumors (n = 14) were randomly divided into blocks of two, with the treatment groups alternating between receiving intratumoral (IT) OMX injection (OMX IT group) and intravenous (IV) OMX injection (OMX IV group). Tumors were regarded as hypoxic if maximum tumor-to-muscle ratio (TMRmax) was greater than 1.4. In addition, hypoxic volume (HV) was defined as the region with tumor-to-muscle ratio greater than 1.4 on [18F]FMISO PET images. Hypoxia was detected in 6/7 tumors in the OMX IT group and 5/7 tumors in the OMX IV injection group. Although there was no significant difference in baseline hypoxia between the OMX IT and IV groups, the two groups showed different responses to OMX. In the OMX IV group, hypoxic tumors (n = 5) exhibited significant reductions in tumor hypoxia, as indicated by decreased TMRmax and HV in [18F]FMISO PET imaging after treatment. In contrast, hypoxic tumors in the OMX IT group (n = 6) displayed a significant increase in [18F]FMISO uptake and variable changes in TMRmax and HV.Conclusions[18F]FMISO PET/CT imaging presents a promising non-invasive procedure for monitoring tumor hypoxia and assessing the efficacy of hypoxia-modulating therapies in canine patients. OMX has shown promising outcomes in reducing tumor hypoxia, especially when administered intravenously, as evident from reductions in both TMRmax and HV in [18F]FMISO PET imaging.

Preparation and bioevaluation of a novel 99mTc-labelled propylene amine oxime (PnAO) containing two 4-methyl-2-nitroimidazole groups as a promising tumor hypoxia imaging agent

Hypoxia is a common phenomenon in solid tumors, and its presence inhibits the efficacy of tumor chemotherapy and radiotherapy. Accurate measurement of hypoxia before tumor treatment is essential. Three propylene amine oxime (PnAO) derivatives with different substituents attached to 2-nitroimidazole were synthesized in the work, they are 3,3,9,9-tetramethyl-1,11-bis(4-bromo-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Br2P2), 3,3,9,9-tetramethyl-1,11-bis(4-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Me2P2) and 3,3,9,9-tetramethyl-1,11-bis(4,5-dimethyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (2Me2P2). The three compounds were radiolabeled with 99mTc to give three complexes([99mTc]Tc-Br2P2, [99mTc]Tc-Me2P2 and [99mTc]Tc-2Me2P2) with good in vitro stability. [99mTc]Tc-Me2P2 with a more suitable reduction potential had the highest hypoxic cellular uptake, compared with [99mTc]Tc-2P2 that have been previously reported, [99mTc]Tc-Br2P2 and [99mTc]Tc-2Me2P2. Biodistribution results in S180 tumor-bearing mice demonstrated that [99mTc]Tc-Me2P2 had the highest tumor-to-muscle (T/M) ratio (12.37 ± 1.16) at 2 h in the four complexes. Autoradiography and immunohistochemical staining results revealed that [99mTc]Tc-Me2P2 specifically targeted tumor hypoxic regions. The SPECT/CT imaging results showed that [99mTc]Tc-Me2P2 could target the tumor site. [99mTc]Tc-Me2P2 may become a potential hypoxia imaging agent.

Towards tumour hypoxia imaging: Incorporating relative oxygen extraction fraction mapping of prostate with multi-parametric quantitative MRI on a 1.5T MR-linac.

Hypoxia plays a central role in tumour radioresistance. Reliable tumour hypoxia imaging would allow the monitoring of tumour response and a more personalized adaptation of radiotherapy planning. Here, we showed a proof of concept of the feasibility and repeatability of relative oxygen extraction fraction (rOEF) mapping of prostate using multi-parametric quantitative MRI (qMRI) achieved for the first time on a 1.5T MR-linac. T2, T2* relaxation times maps, and intra-voxel incoherent motion (IVIM) parametric maps mapping were computed on a 29 years old healthy volunteer. R2' and rOEF maps werecalculated based on a multi-parametric model. Long-term repeatability and repeatability coefficient (RC) were determined for each parameter according to QIBA recommendations. Mean values for the entire healthy prostate were 0.99 ± 0.14 × 10-3 mm/s2, 81 ± 2.1 × 10-3 mm/s2, 21.6 ± 3.6%, 92.7 ± 19.7 ms and 62.4 ± 17.3 ms for Dslow, Dfast, f, T2 and T2*, respectively. R2' and rOEF in the prostate were 6.1 ± 3.4 s-1 and 18.2 ± 10.1% respectively. The RC of rOEF was 4.43%. Long-term repeatability of quantitative parameters based on a test-retest ranged from 2 to 18%. qMRI parameters are measurable and repeatable on 1.5T MR LINAC. From T2, T2* and IVIM parameters maps, we were able to obtain a rOEF mapping of the prostate.These results are the first step to a non-invasive imaging of tumour hypoxia during radiotherapy leading to a biological image-guided adaptive radiotherapy.

A Contrast-Enhanced Tri-Modal MRI Technique for High-Performance Hypoxia Imaging of Breast Cancer.

Personalized radiotherapy strategies enabled by the construction of hypoxia-guided biological target volumes (BTVs) can overcome hypoxia-induced radioresistance by delivering high-dose radiotherapy to targeted hypoxic areas of the tumor. However, the construction of hypoxia-guided BTVs is difficult owing to lack of precise visualization of hypoxic areas. This study synthesizes a hypoxia-responsive T1, T2, T2 mapping tri-modal MRI molecular nanoprobe (SPION@ND) and provides precise imaging of hypoxic tumor areas by utilizing the advantageous features of tri-modal magnetic resonance imaging (MRI). SPION@ND exhibits hypoxia-triggered dispersion-aggregation structural transformation. Dispersed SPION@ND can be used for routine clinical BTV construction using T1-contrast MRI. Conversely, aggregated SPION@ND can be used for tumor hypoxia imaging assessment using T2-contrast MRI. Moreover, by introducing T2 mapping, this work designs a novel method (adjustable threshold-based hypoxia assessment) for the precise assessment of tumor hypoxia confidence area and hypoxia level. Eventually this work successfully obtains hypoxia tumor target and accurates hypoxia tumor target, and achieves a one-stop hypoxia-guided BTV construction. Compared to the positron emission tomography-based hypoxia assessment, SPION@ND provides a new method that allows safe and convenient imaging of hypoxic tumor areas in clinical settings.

Establishment and Application of Novel Hypoxia-driven Dual-reporter Model to Investigate Hypoxic Impact on Radiation Sensitivity in Human Nasopharyngeal Carcinoma Xenografts.

Background: Tumor hypoxia has been frequently detected in nasopharyngeal carcinoma (NPC) and is intently associated with therapeutic resistance. The aim of the study is to establish a clonogenically stable hypoxia-inducible dual reporter model and apply it to investigate the effect of tumor hypoxia on DNA double strand break (DSB) and synergistic effect of irradiation in combination with chemotherapy or targeted therapy. Methods: The plasmid vector consisting of hypoxia response elements to regulate HSV1-TK and GFP genes, was constructed and stably transfected into human NPC cells. The expected clone was identified and validated by in vivo and in vitro assay. DSB repair was measured by γH2AX foci formation. Tumor growth delay assay and spatial biodistribution of various biomarkers was designed to investigate the anti-tumor effect. Results: The system has the propensity of high expression of reporter genes under hypoxia and low to no expression under normoxia. Intratumoral biodistributions of GFP and classic hypoxic biomarkers were identical in poor-perfused region. Upon equilibration with 10% O2, the xenografts showed higher expression of hypoxic biomarkers. Cisplatin radiosensitized SUNE-1/HRE cells under hypoxia by suppressing DSB repair while the addition of PI3K/mTOR inhibitor further enhanced the anti-tumoral therapeutic efficacy. Combination of IR, DDP and NVP-BEZ235 exhibited most effective anti-tumor response in vivo. These observations underline the importance of dual reporter model for imaging tumor hypoxia in therapeutic study. Conclusions: Our preclinical model enables the investigation of heterogeneous tumor hypoxic regions in xenograft tissues and explores the treatment efficacy of combinations of various therapeutic approaches to overcome hypoxia.

Intracellular Nitroreductase-Triggered "On" and "Enhanced" Photoacoustic Signals for Sensitive Imaging of Tumor Hypoxia.

Current molecular photoacoustic (PA) probes are designed with either stimulus-turned "on" or assembly-enhanced signals to trace biological analytes/events. PA probes based on the nature-derived click reaction between 2-cyano-6-aminobenzothiazole (CBT) and cysteine (Cys) (i.e., CBT-Cys click reaction) possess both "turn-on" and "enhanced" PA signals; and thus, should have higher sensitivity. Nevertheless, such PA probes, particularly those for sensitive imaging of tumor hypoxia, remain scarce. Herein, a PA probe NI-Cys(StBu)-Dap(IR780)-CBT (NI-C-CBT) is rationally designed, which after being internalized by hypoxic tumor cells, is cleaved by nitroreductase under the reduction condition to yield cyclic dimer C-CBT-Dimer to turn the PA signal "ON" and subsequently assembled into nanoparticles C-CBT-NPs with additionally enhanced PA signal ("Enhanced"). NI-C-CBT exhibits 1.7-fold "ON" and 3.2-fold overall "Enhanced" PA signals in vitro. Moreover, it provides 1.9-fold and 2.8-fold overall enhanced PA signals for tumor hypoxia imaging in HeLa cells and HeLa tumor-bearing mice, respectively. This strategy is expected to be widely applied to design more "smart" PA probes for sensitive imaging of important biological events in vivo in near future.

A Novel Tumor Hypoxia Imaging Agent: [99mTc]Tc(CO)3-CPA-2-NIM.

Hypoxia imaging agents can selectively remain in hypoxic tissue, which can directly reflect the location and degree of hypoxia. Synthesized a novel tumor hypoxia imaging probe [99mTc]Tc(CO)3-CPA-2-NIM and evaluated its biological behavior with the purpose to assess its possibility of becoming a qualified tumor hypoxia imaging agent. Radiochemcial purity of [99mTc]Tc(CO)3-CPA-2-NIM was greater than 95% after HPLC purification. Lipophilicity coefficient of this complex was -1.74 ± 0.10 (n = 5, number of experiments), indicating it was a hydrophilic complex. In vitro cell experiments demonstrated that this complex has selectivity for hypoxia at oxygen concentrations < 10 ppm (parts per million). Biodistribution experiment in S180 tumor bearing mice showed that tumor uptake reached its highest at 2 h post-injection with mice tumor-to-muscle ratio. Complex [99mTc]Tc(CO)3-CPA-2-NIM has the possibility of becoming a tumor hypoxia imaging agent.

Synthesis and Evaluation of 99mTc-Labelled 2-Nitroimidazole Derivatives with Different Linkers for Tumour Hypoxia Imaging.

When developing novel radiopharmaceuticals, a linker moiety between the chelator and targeting vector can have a crucial influence on adjusting the affinity of the tracer and its biodistribution in organisms. To develop novel 99mTc-labelled hypoxia imaging radiotracers, in this study, five isocyanide-containing 2-nitroimidazole derivatives with different linkers (L1, L2, L3, L4 and L5) were synthesised and radiolabelled with technetium-99m to obtain five stable 99mTc-complexes ([99mTc]Tc-L1, [99mTc]Tc-L2, [99mTc]Tc-L3, [99mTc]Tc-L4 and [99mTc]Tc-L5). Corresponding rhenium analogues of [99mTc]Tc-L1 were synthesised and suggested the structures of these 99mTc-complexes would be a monovalent cation with a technetium (I) core surrounded by six ligands. [99mTc]Tc-L1 is hydrophilic, while the lipophilicities of [99mTc]Tc-L2, [99mTc]Tc-L3, [99mTc]Tc-L4 and [99mTc]Tc-L5 are close. In vitro cell experiments showed that all five novel 99mTc-complexes had higher uptake in hypoxic cells compared with aerobic cells, which indicates the complexes have good hypoxia selectivity. The biodistribution of the five 99mTc-complexes in S180 tumour-bearing mice showed that they all had certain uptake in the tumours. Among them, [99mTc]Tc-L1 had the highest tumour-to-muscle (4.68 ± 0.44) and tumour-to-blood (3.81 ± 0.46) ratios. The introduction of polyethylene glycol (PEG) chains effectively reduced the lipophilicity and decreased uptake by the liver, intestine and blood but also increased clearance from the tumours. In vivo metabolic studies showed [99mTc]Tc-L1 kept intact and remained stable in tumour, blood and urine at 2 h post-injection. The results of SPECT imaging showed that [99mTc]Tc-L1 had significant tumour uptake at 2 h post-injection, but there was still high uptake in abdominal organs such as the liver and kidney, suggesting that this complex needs to be further optimised before being used for tumour hypoxia imaging.

In Vivo Imaging of Tumor Hypoxia by Maintaining Green Fluorescence of 9-Aminoanthracene Under Hypoxic Conditions

In this study, 9-aminoanthracene (9AA) was used as a new fluorescence reagent for the in vivo imaging of tumor hypoxia by taking advantage of the maintenance of its green fluorescence under hypoxic conditions. As 9AA is insoluble in water, polyethylene glycol (PEG)-400 was used to dissolve 9AA in saline. Each organ was successfully stained with 9AA, as observed by green fluorescence using in vivo imaging, following intragastric administration of a 9AA PEG-saline solution in mice. Therefore, the intragastric administration of 9AA can be used for in vivo imaging of normal mice. Tumor hypoxia staining using the 9AA fluorescence method was evaluated by in vivo imaging of mice subcutaneously transplanted with Ehrlich ascites carcinoma cells and compared with conventional pimonidazole (PIMO) staining under hypoxic conditions. The tumor sections were stained with green fluorescence derived from 9AA and the same sections corresponded to hypoxic areas upon immunohistochemical staining with PIMO.

Advances in PET and MRI imaging of tumor hypoxia.

Tumor hypoxia is a complex and evolving phenomenon both in time and space. Molecular imaging allows to approach these variations, but the tracers used have their own limitations. PET imaging has the disadvantage of low resolution and must take into account molecular biodistribution, but has the advantage of high targeting accuracy. The relationship between the signal in MRI imaging and oxygen is complex but hopefully it would lead to the detection of truly oxygen-depleted tissue. Different ways of imaging hypoxia are discussed in this review, with nuclear medicine tracers such as [18F]-FMISO, [18F]-FAZA, or [64Cu]-ATSM but also with MRI techniques such as perfusion imaging, diffusion MRI or oxygen-enhanced MRI. Hypoxia is a pejorative factor regarding aggressiveness, tumor dissemination and resistance to treatments. Therefore, having accurate tools is particularly important.

Afterglow/Fluorescence Dual-Emissive Ratiometric Oxygen Probe for Tumor Hypoxia Imaging

Hypoxia is a common feature of many diseases such as solid tumors. The measurement and imaging of oxygen (O2) are extremely important for disease diagnosis and therapy evaluation. In this work, the afterglow/fluorescence dual-emissive ratiometric O2 probe based on a photochemical reaction-based afterglow system is reported. The afterglow is highly sensitive to O2 because the O2 content is directly related to the 1O2 yield and eventually affects the afterglow intensity. The O2-insensitive fluorescence of an emitter can serve as an internal reference. As the O2 concentration changes from 0.08 to 18.5 mg L-1, the ratio value shows a remarkable 53-fold increase. Compared with the intensity of a single peak, the ratiometric signal can eliminate the interference of the probe concentration to achieve higher accuracy. This afterglow/fluorescence dual-emissive ratiometric O2 probe is successfully applied to hypoxia imaging in tumor-bearing mice, which may further promote the development of O2 sensing in the biomedical field.

Bioparameter-directed nanoformulations as MRI CAs enable the specific visualization of hypoxic tumour.

A malignant tumour has hypoxic cells of varying degrees. The more severe the hypoxic degree, the more difficult the prognosis of the tumour and the higher the recurrence rate. Therefore, tumour hypoxia imaging is crucial. Magnetic resonance imaging (MRI) shows its strength in high resolution, depth of penetration and noninvasiveness. However, it needs more excellent contrast agents (CAs) to combat the complex tumour microenvironment (TME) and increased targeting of tumours to enhance clinical safety. Many research studies have focused on developing hypoxia-responsive MRI CAs that take advantage of the unique characteristics of hypoxic tumours. The low oxygen pressure, acidic TME, and up-regulated redox molecule levels found in hypoxic tumours serve as biological stimuli for nanoformulations that can accurately image the hypoxic region. This review highlights the importance of developing bioparameter-directed nanoformulations as MRI CAs for accurate tumour diagnosis. The design strategies and mechanisms of tumour-hypoxia imaging with nanoformulations are exemplified, with a focus on pH-responsiveness, redox-responsiveness, and p(O2)-responsiveness. The promising future of bioparameter-responsive nanoformulations for accurate tumour diagnosis and personalised cancer treatment is discussed.

Nitroreductase-sensitive fluorescent covalent organic framework for tumor hypoxia imaging in cells.

Covalent organic frameworks (COFs) have been used in cell imaging, but very rarely for imaging specific cell conditions. Herein, a β-ketoenamine-based fluorescent COF was post-synthetically modified to incorporate a hypoxia-targeting molecule. Fluorescence microscopy imaging shows that the material discriminates between HeLa cells grown under hypoxia and those cultured under normoxia.

Enhancing NIR-II Phosphorescence through Phosphorescence Resonance Energy Transfer for Tumor-Hypoxia Imaging

Enhancing NIR-II Phosphorescence through Phosphorescence Resonance Energy Transfer for Tumor-Hypoxia Imaging

Photoacoustic imaging a PDT response marker for monitoring vasculature changes

Photoacoustic imaging (PAI) is an evolving modality for studying and imaging tumor hypoxia. The experimental results from animals treated with ALA for Pre-PDT, Post-PDT, 4hr PDT, 24 hr PDT show a short-term increase in the BV followed by a strong decrease; however, no complete stasis. An alternate method is created by looking at the spatial and frequency domain histogram to observe the tumor vasculature. The images are represented in frequency domains by application of two-dimensional Fast Fourier Transform (FFT) on spatial domain images. The variations in acoustic impedance after Photodynamic Therapy (PDT) are studied. The photoacoustic signals implemented on tumor models indicated PDT influenced variations in the hemoglobin levels against different time periods and studied their outcome on the tumor necrosis.