Tumour Microenvironment
Ralph Peter Mason1

1Radiology, University of Texas Southwestern Medical Center, Dallas, TX, United States

Synopsis

Historically, radiology/imaging has served to identify tumors in terms of location, size, and metastatic spread. It is increasingly recognized that tumors may exhibit very different micro environmental characteristics, which can influence therapeutic success. A new goal is precision oncology, whereby individual tumors are further characterized based on potential prognostic imagine biomarkers. Tumor hypoxia is associated with aggressive phenotypes and resistance to therapy and may be the most significant factor influencing therapy outcomes for solid tumors. Many NMR approaches are being developed and evaluated to measure tumor oxygenation. This review will consider human applications of oxygen sensitive MRI in the context of pre-clinical developments. Strengths and weaknesses in terms of temporal and spatial resolution, precision and accuracy, ease of implementation and robustness of observations will be considered. Methods may provide qualitative or quantitative insights including dynamic response to interventions.

Highlights

1. Tumor hypoxia is associated with aggressive phenotypes, notably influencing angiogenesis, metastasis, and response to therapy. Therefore, the ability to identify hypoxia could allow stratification for optimized therapy.

2. MRI methods have been developed to quantify pO2, and hence tumor hypoxia, or provide pertinent surrogate biomarkers.

3. This presentation will consider opportunities and applications for patients in the context of competing technologies.

4. Virtues and shortcomings of diverse techniques will be considered in terms of ease of implementation, nature of observations (spatial resolution, precision, dynamics, and need for exogenous reporter agents) and validity and robustness of measurements.

5. Examples will be drawn from recent studies in human, focusing on our own experience.

Target audience – Imaging scientists/radiologists, oncologists, physiologists, and scientists interested in tumor oxygenation.

Context Background

Historically, radiology has served to identify tumors in terms of location, size and metastatic spread. Specific acquisition sequences such as T2-weighting may enhance tumor delineation. Contrast agents enhance sensitivity and specificity based on relative vascular permeability and agent retention. Metastatic spread is probably best revealed by FDG-PET. A new paradigm is personalized oncology based on the unique characteristics of the individual tumors. Crucial goals are predicting the optimal therapy and providing early indications of therapeutic efficacy. Several parameters are considered pertinent, since they seem to differ in tumors versus normal tissue: notably, vascular perfusion, pH and oxygenation. Tumors are often characterized by convoluted, disorganized vascular networks providing inefficient blood flow and nutrient supply. The direct consequence is often hypoxia and this may lead to elevated glycolysis, and hence, acidosis. These parameters are interrelated and may provide insights into tumor aggressiveness and optimal therapy.

The most widespread assessment of tumor pathophysiology has been based DCE-MRI with thousands of studies reported using various levels of analytical sophistication (1-3). Dynamic contrast curves have been related to the stage of breast cancer, though there remains discussion of the optimal technique with trade-offs between spatial and temporal resolution and quantitative versus qualitative analysis (4-6). In prostate, indications of texture analysis based on differential enhancement curves appear valuable to delineate tumors and integration of multiparametric imaging is showing promise (7-9). Several studies have indicated the DCE-MRI may be related tumor hypoxia, e.g., correlations with polarographic oxygen electrodes in human cervical cancer (10). Various supporting studies have been reported in diverse melanoma and tumor models by the group of Rofstad (11, 12). We recently found the strong correlation between the parameter ve and time for rat prostate tumors to quadruple in volume following single high-dose radiation (essentially SBRT) (13). DCE-MRI is attractive, since it is routine in the clinic, but there is a major emphasis on developing methods more directly related to tumor oxygenation.

Oxygen Enhanced MRI: TOLD

The oxygen molecule is paramagnetic and enhances spin-lattice relaxation (14). In saline R1=f(pO2). It has been exploited to measure pO2 in various human body fluids in situ, where the composition is well-characterized and homogenous, e.g., urine (15), CSF (16) and vitreous humor (17). Other factors such as ions, protein concentration, and pH can influence R1 and it is not expected to directly to reflect pO2 in tissues in vivo. However, a change in R1 accompanying an intervention such as hyperoxic gas challenge should reflect a variation in pO2 (ΔR1=f(ΔpO2)). Several investigations have demonstrated such changes in both pre-clinical and human research studies. Many studies have examined changes in T1-weighted signal reflecting a variation in R1, but increasingly quantitative R1 maps are presented. In 1996, Edelman et al., reported oxygen enhanced MRI of human lung in response to an oxygen breathing challenge and they observed differential regional ventilation (18). They noted that the relaxivity of oxygen is small, but found substantial changes attributed to the large alveolar surface area. Super oxygen-saturated water has been explored for GI contrast (19).

Tumors are more complex and it should be noted that changes in hemoglobin concentration and pH can influence R1 and flow may alter apparent R1. Howe et al., noted T1-weighted signal response in tumors accompanying a carbogen breathing challenge (20). However, the changes were generally small and subtle, compared to BOLD response (discussed below) and were therefore largely neglected. In 2001, Matsumoto et al., showed a significant response in R1 to hyperbaric oxygen challenge in tumors and suggested the acronym TOLD (tissue oxygen level dependent) contrast by analogy to BOLD (21). This work ignited interest from several groups. Notably, O’Connor et al. explored both human applications and pre-clinical tests (22-26). They demonstrated R1 response in normal human organs in response to oxygen breathing challenge and in pre-clinical studies of human tumors. Correlations were determined with hypoxia based on immunohistochemistry (27). Most recently, they showed that correlation of R1 and pimonidazole defined hypoxia was improved in some tumor types by including a DCE-MRI scan to define non-perfused regions (22).

Garbow et al., recently presented correlations of R1 and pO2 based on electrodes in assessment of rat brain tumors with respect to oxygen breathing challenge (28). Notably, they were able to differentiate tumor from radiation necrosis, which could become very valuable in man. Remmele et al. (29, 30), reported several studies in human brain and tumors, generally in conjunction with R2*, as described below. Likewise, we generally undertake combined BOLD and TOLD studies (31, 32) (vide infra). Unger et al., have applied TOLD to evaluate essential changes in brain tumor oxygenation with respect to irradiation and administration of a perfluorocarbon emulsion designed to mitigate hypoxia (33). The study to be presented in Session: Interventional 1, 5 PM Tuesday, May 10, 2016, indeed shows shortened T1 in brain tumors following irradiation and PFC administration consistent with improved oxygenation.

We have examined TOLD extensively in Dunning prostate rat tumors and found responses consistent with BOLD and quantitative 19F MRI oximetry in many tumors (Fig. 1) (31, 32). Moreover, we found that tumor growth delay (time to quadruple in volume, T4) following single high dose irradiation (30 Gray) was related to TOLD response (but not BOLD) (31). Specifically, those tumors showing a large TOLD (ΔSI or ΔR1) response to an oxygen breathing challenge prior to irradiation, showed a significantly greater tumor growth delay (Fig. 1). In common with most pre-clinical investigations, our initial investigations examined subcutaneous tumors, but we have recently demonstrated TOLD in orthotopic rat prostate tumors also (Fig. 1E) and have recently demonstrated R1 response to oxygen breathing challenge in human prostate cancer. Oxygen challenge was added to the standard pre-surgical treatment planning and compliance was excellent.

Oxygen Enhanced MRI: BOLD

To date, BOLD assessment of tumors has been far more popular with human studies presented at several disease sites (34) including breast (Fig. 2) (35, 36), cervix (37, 38) (Fig. 3), prostate (39, 40), kidney (41, 42), head and neck (43), brain (44, 45) and lung (Fig. 4). R2* is strongly influenced by the concentration of deoxyhemoglobin, as exploited in fMRI of neuronal activation (46). Extensive studies from Griffiths, Howe, Robinson et al. showed differential BOLD response to oxygen breathing challenge and the magnitude could be related to vascular extent (20, 47-50). Many studies have used semi-quantitative measurements based on %ΔSI though this may be influenced by flow effects, as presented in the FLOOD concept (20). Several studies have examined correlations of quantitative oximetry (pO2) with BOLD and general trends are widely reported, but the magnitude of response may be highly variable (51-53). We found that a BOLD signal response to oxygen breathing challenge greater than 3% in 13762NF rat breast tumors coincided with elimination of hypoxia (52). However, specific inconsistencies have been noted, e.g., increased BOLD signal following sacrifice has been attributed to pressure induced vascular collapse post mortem and hence decreased concentration of deoxyhemoglobin (47). Gallez et al., also reported discrepancies with respect to pharmacological interventions (54).

Many human investigations have focused on proof of principle demonstrating that BOLD is feasible in human tumors. Baseline R2* values were related to human prostate tumor hypoxia (55). Rijpkema el al. examined BOLD changes in head and neck tumors in connection with the ARCON therapeutic trial (43). We found that women whose breast tumors showed a large BOLD response to oxygen breathing challenge prior to therapy had a complete radiological and pathological response following adjutant chemotherapy for locally advanced breast cancer whereas those with small BOLD showed at best stable disease (35) (Fig. 2).

Combining BOLD and TOLD

As noted, BOLD and TOLD have been related to tumor oxygenation. We favor combining the approaches particularly for the assessment of tumor oxygenation with respect to respiratory gas challenge and have proposed the acronym DOCENT (Dynamic Oxygen Challenge evaluated by NMR T1 and T2*) (56). Specifically, BOLD is sensitive to vascular delivery, but does not necessarily indicate change in pO2. Meanwhile, TOLD is more closely related to pO2, but the magnitude of response tends to be much smaller (Fig. 1). A dynamic combination is technically feasible emphasizing the larger BOLD effect confirmed by TOLD. As expected, TOLD response is usually temporarily delayed compared with BOLD, since oxygen is first delivered in the vasculature and must then diffuse into tissues. In both pre-clinical and human studies, we generally perform a baseline R1 map followed by interleaved R2* and T1-weighted images to reveal dynamic changes and finally another R1 map.

In both Dunning prostate R3327-AT1 and -HI tumors, we found a close correlation between semi-quantitative T1- and T2*-weighted signal response to oxygen breathing challenge, but BOLD was generally three times greater (32). Intriguingly, while the well vascularized HI tumors also showed a correlation between mean ΔR1 and ΔR2*, this was noticeably absent for AT1 tumors (32). Voxel-by-voxel analysis showed very diverse responses including matched and unmatched behavior. Remmele et al., similarly reported matched and unmatched responses in human brain tumors and proposed the following rationale (29). In regions that are well oxygenated, excess dissolved oxygen will increase the plasma and tissue fluid R1, but will have negligible effect on hemoglobin (Hb) saturation (hence, small effect on R2*). Meanwhile, hypoxic regions with suboptimal oxygen saturation will show preferential binding of excess oxygen molecules to deoxygenated Hb molecules, which reduces the content of paramagnetic deoxyHb, shortening both R2* and R1. In the absence of deoxyHb (e.g., fluids like in cystic tumor regions or edema), the excess oxygen in solution prolongs both R1 and R2*. Non-oxygenation-related R2* increase and R1 decrease, potentially related to vascular steal.

Comparison of BOLD and TOLD effects in tumors has been reported for various species including mice (57, 58), rats (31, 32), rabbits (59) and man (24, 30). O’Connor et al. noted distinctly different BOLD and TOLD effects in certain normal organs, as we have also observed (Fig. 5) (24, 30, 60).

MOBILE

Recognizing the greater solubility of oxygen in lipids as compared with water, Gallez et al. recently proposed MOBILE (Mapping of Oxygen By Imaging Lipids relaxation Enhancement) (61). They specifically showed that changes in lipid relaxation were greater than water: about 2 and 11 fold greater for the resonances at 1.2 and 4 ppm, respectively. Observations were reported in mice, with respect to ischemia, liver steatosis, and tumors (62). This method requires effective water suppression, but pilot studies have also been reported in human volunteers (63).

MOXI

Recently, Zhang et al. (64) demonstrated that a multi parametric analysis of tumor water signal could directly provide estimates of pO2. Specifically, biexponential analysis of IVIM (Intra Voxel Incoherent Motion) allowed estimation of tumor vascular volume; then in combination with R1 and biexponential analysis of CPMG-based R2, yielding the extravascular component, an accurate measurement of pO2 was achieved, as validated using 19F MRI of PFC. The current measurements are limited to air breathing due to model assumptions, but can provide an effective quantitative baseline maps from which to evaluate BOLD and TOLD responses to interventions. To date measurements have been limited to rat tumors and calculations will need to be modified for other species due to differences in hematocrit and oxygen-hemoglobin binding constants.

Summary

Oncologists and patients increasingly have choice of many potentially effective therapies. However, the optimal intervention may depend on unique characteristics of a specific tumor; hence the desire for precision oncology. Hypoxia has a significant role in tumor aggressiveness and response to therapy and therefore the ability to measure tumor oxygenation and dynamic response interventions could play a significant role in treatment. Hypoxia may justify a radiation boost potentially with local dose painting based on intensity modulated radiotherapy or selection of alternant ionizing beams, such as proton or carbon and inclusion of hypoxic cell selective cytotoxins. As presented, there are several NMR approaches to assessing tumor oxygenation. Examples have been demonstrated in pre-clinical models and increasingly are being evaluated for effective translation to humans, but it remains to be seen which will be most effective in terms of robust prognostic imaging biomarkers offering useful sensitivity and specificity. Further relevant discussion is provided in the companion tutorial on Tumor Hypoxia Imaging (#7138).

Acknowledgements

Research presented in this tutorial is currently supported by CPRIT RP120753-03, RP140285, RP140399 and previously DOD and NIH.

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Figures

Figure 1 Tumor oxygenation and radiation response. A) BOLD, TOLD, %ΔT2* for tumors exhibiting diverse response to O2-challenge, B) Corresponding pO2, C) Radiation response (volume quadrupling time T4) and D) Dependence on ΔT1 for O2 group (31), E) BOLD and TOLD for orthotopic rat prostate tumor (unpublished data from Dr. White).

Figure 2 Comparison of pre-chemotherapy BOLD MRI enhancement of breast tumors with respect to treatment outcome (35). Images show representative enhancement maps pretherapy for a tumor ultimately showing complete pathological response (CR) versus stable disease (right). Graphs shows mean BOLD MRI enhancement pre-chemotherapy of each tumor with oxygen challenge.

Figure 3 BOLD response to oxygen challenge in cervical cancer. (a) T2w image showing cervical tumor (T); (b) T2*w image showing (ROIs); R2* maps with (C) air and (D) oxygen breathing; (e, f) ROI-based T2*w signal decay measurements; (g) Mean R2*; h) relative signal at TE=39ms in MGRE (37).

Figure 4 BOLD MRI of patient with lung tumor A) R2* measurements: SAG 3D SPGR (6-echo; TE1=1.24ms; ΔTE=5ms; TR=28ms); B) derived R2* map while breathing air; C) R2* distributions in tumor; D) significant change was observed in median R2* upon breathing oxygen. Unpublished data with Drs. Yuan, Timmerman and Pedrosa.

Figure 5 Interleaved R1 and R2* in a healthy volunteer (60). T1w image (A) and T2*w images (D) show normal anatomy of abdominal organs. (B) R1 map with air; (C) R1 in spleen and renal cortex showed significant increase with O2 breathing. Corresponding R2* maps show little change.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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